Growth and involution of lymphatic tissue and their relation to secretions of the adrenal cortex in CBA mice

The primary aims of these experiments were: (1) to determine the responses of various representative lymphatic organs to the process of aging in intact animals, (2) to determine the responses of the various lymphatic organs to adrenalectomy at successive ages. From the data obtained it was hoped to secure information on the role of the adrenal glands in the control of growth and involution of each of the lymphatic organs. Studies were made of the normal body growth and of the changes in absolute and relative weights of lymphatic and certain non-lymphatic organs from prepuberty, through puberty and sexual maturity to senility in male and female mice of the CBA strain. These studies of the effect of adrenalectomy on the growth and involution of the lymphatic organs have shown that: 1. The secretions of the adrenal glands, probably the cortex, restrain growth and enhance involution. 2. The influence of the adrenal glands is greatest in the youngest animals and in the oldest animals, indicating greater secretory activities of these glands during those periods of life. 3. The lack of the secretions of the adrenal glands, however, does not permit unlimited growth. The increased growth following adrenalectomy is greatest after a five day period. 4. The different lymphatic organs respond differently to adrenalectomy, although all except the thymus following the same general pattern. The effects on the thymus are, in general, the reverse of those of other organs. 5. Each lymph organ responds differently at different ages. In all but the thymus the response tends to be greatest in the young animals and senile animals, and least in the sexually mature animals. The response of the thymus tends to be greatest in the sexually mature animals. 6. There exist sex differences in the response of all lymphatic organs to adrenalectomy at all ages. There are small and more quantitative in nature and are most evident in young animals. These sex differences indicate a role played by sex hormones in controlling growth and involution of lymphatic tissue. 7. The weights of the adrenal glands were greater in the females at all ages and this may be correlated with the greater relative weights of lymphatic organs of the females. 8. These variations among glands and in age, sex, and period following adrenalectomy must be recognized in comparing data obtained from the literature and in future experiments.

TFrn: GROWTH AND INVOLU'l'ION OF LYMPHATIC TISSUE AND THEIR RELArrION TO SECRETIONS OF THE ADRENAL CORTEX IN CBA MICE by George A. Santisteban A thesis submitted to the faoulty of the University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy .Department of Ana tomy Approved: utah UNIVEl~U -; TY OF UTiIlI LIBRARYi PREFACE In 1949, Dr. Thomas F. Dougherty assigned this author the task or translating Dr. Carlos E. Rapela 1 s monograph on the relattonships between the a dl" ena 1 gland and the tt~US, also Dr. Hugo Chlodl's monograph. on the relatlonship between the sex;;lands and the thymus. These two pleces of work aroused in me an interest in the interrelationships between the endoorine systefllsnd the growth llnd involution of lymphatic tissue. I am f::;reatly indebted to Dr. Thomas F'. Dougherty, who encouraged me and who made available the facilities for making this investIgation. I wish to express my gratitude to til"'. Gottlieb Schneebeli for his rnany helpful sug;:~est1ons Juring the conducting of the experiments, to Eiss Kathryn Seymour for her technical assis­tance, and to Mrs. BarI)a.ra Santlstaban for the typing of the manuscript. I nm grateful to Dr. '1'. F. Dou~,:her tY' and to :eX'. Kenneth R. Brizzee for their helpful ~mgr~estlons 1n the preparation of' tl1c manuscrIpt. rro Dr. W. 'Ii. NewbY' r ortle a. special word of than};:s for his kind and pationt 8.ss1staneein the final reading of the manuscript and for his many helpful cr1 tic1sr.::s. 285657 11 TABLE OF OONTENTS Page PREFACE • • • • • • • • • • • • • • • • • • • • • • 11 LIST OF TABLES • • • • • • • • • .. • • .. • • .. ... v1 LLT OF ILLUSTRATIONS .. • • • • .. • .. .. • .'. '! • • viii Chapter I. II. INTRODUCTIO~l • • • • . .. • • • • .. . • • • RFVIt:~W OF LITERATURE • .. .. .. . • • • • • • A. B. The Normal Growth of Lymphatic Tissue The Normal Growth of Lyrapha tic Tissue and the Adrenal Gland .. • • • .. .. • • .' .. • • .. . • • 1 7 7 12 III. EXPOSITION A. Scope and Purpose or Study • • .. .. • • •• 24 B. Materials and General Methods • .. • • •• 2e C. Discussion and Definition of Terms • • ... 28 IV. HT~SUL'I'8 O.li' EXPERIVENTS A. The NOrma 1 Body Growth and the Growth of Lymphatic and Non-lymphatic Organs of Two to Twenty-two Weeks of Age 1. Procedure .. 11 . . .. . .. . . • • • • • • ss 2. Results Bod7 Weight • • • .. • • .. .. .. • • .. .... 39 Lymphatic and endocrine organs .. .. •• 40 Inguinal and axillary nodes .. •• .. ... 41 Mesenteric Nodes .. • .. • • .. • • • •• 44 Thyml .. .. • • • • • • • • • • • • .. •• 44 Spleens .. .. • .. • • • • • .. • .. • • •• 46 Adrenals • • • • • • • • • • • • .. •• 48 Testes • .. • • .. • • • • • • • • • •• 51 3. Summary and Discussion • • • • • • •• 57 111 Page B. Chan:3ea in Body Weights and in Wei.~hts of Lymphatic and Non-l)T.1phatlc Or:.:;ans of CP,A Mice Four Days After Adrenalectomy 1. Introduction. • • • • • • • • • • • •• 57 2. Methods. • • • • • • • • • • • • • • •• 57 3. Sesults Body Weight • • • • • • • • • • • • • •• 56 Lymphs. tic oI',,;e.ns and kidney • • • • • • • 60 In,7uinnl and axillary nodes • • • • • •• 50 Mesenterio nodes • • • • • • • • • • •• 65 'I'hyl'l1uS • • • • • • • • • • • • • • • •• 67 Spleen • • • • • • • • • • • • • • • •• 67 4. Stunmary and Dlscussi·n " • • • • • • •• 70 G. rrhe 110rmal Growth of Lymph Nodes, r.f'hyrnus, Spleen, Genital Tissues Dnd Adrenal Glands in t,;tce fro:n Six to Sixty-two Weeks of Ar~e • 1. Introduction • • • • • • • • • • • • • • 74 2. Procedure • • • • • • • • • • • • • • • 74 3. Results J'eneral Body Gpowth • • • • • • • • • • • 79 Growth 0 f the testes • • • • • • • • •• 80 Growth of the cva"!'y and uterine tube •• 80 Growth of the adrenal ,~lands • • • • •• 82 Growth and involution of the total lympbs. tic organs • • .. • • • • • • •• H4 GJl'owth and involution at the total bodes 86 Growth and involution of the mesenteric nodes • • • • • .. • • • .. • • • • •• 89 Growth and involution of the peri?~hernl nodes • • • • • • • .. • • • • • • Growth and involution of the spleen Growth and invC'"lution of the thymus Normal A:"fJ chan-'os in proportion of spleen, th~us and l;!r:,nph :::rGC"es to total l)"mphl1 tic o!'gans • • .. • • • • • • • • . .. DIscussion ' • • • • • • • • • • • • • ~o 91 93 96 100 5. SU:r1mal'Y. • • • • .. • • • .. • • • •• 107 iv VI. Page D. Alterations in Body Weight and in Wei~hts of Lymphatio and Genital Organs at Cno, Five, emu Ten Days After Adrenal­eo tom)" 1n egA Mice of Prepubertal to Soni16 Ali'GS t:) 1. Introduc tlc,ll . . . . .. .. . . .. .. . .. . . Procedure . .. .. . .. . .. . .. . .. . .. . .. 110 110 3. R.esults B()c.y 'l1ei-':ht .. • • • • .. .. • • •• • • .. 111 GEl:l! tal t1ssue .. • .. .. .. .. • .. • • .... 116 /llt.eT'stions in absolute and relative weights of lymphatIc organs .. .. .. ... 116 'I'ctal lytn;-hatic or:~t.n.,c ...... OJ .. •• 118 Total nodes .. .. .. .. • .. .. .. • • • • ... 124 F'er:tph.eral nodes .. .. OJ • .. .. • • • ... 129 Mesenteric nodes. .. • •• • .. • .. • •• 134 Sploen .. .. • •• • .. • .. • • .. • • •• 1:38 Thymus .. .. .. .. .. .. .. .. • .. • • .. • .... 145 4. Summary and Disoussion • .. • • .. .. •• 150 LI'l'ert.A':rUR.E CITeD • • .. • • .. • • • • • • • • • 164 LIST OF TABLE:S Table 1. Normal Age Changes in Body Weights and Abso­lU. te Wetghts of Lymphatic and lion-Lym')ha tlc Page Or~ans of Male aDA Mice • • • • • • • • • • •• 34 2. Normal Age Changes in Body Wel:hts a.nd Abso­lute Vlei';hts of Lymphs tic and Non-L;:,rnlpha tic Organs of Pemale Ct1A 1111oe.. • • • • • • • • •• 35 3. Normal Age Changes in Relative Weights of Lyr:q)hatlc and Non-Lymphatic Orga.ns of Male CBA Mice . • • • • • • • • • • • • • • • • • • • •• 37 4. Normal Age Changes in Relative Wei~lhts of L'j'11lphatic and Non-Lyrn~)ha.tic Organs of Female CBA Mice • • • • • • • • • • • • • • •• • • 5. Losses in Body Weights of Male and Female CBA • • 38 Mice Four Days After Adrenalectomy • • • • • •• 58 6. Average Body & Organ Weights in Female CBA ~~l1ce, Intact and at Fbur Days After Adrenalectomy •• 61 7. Average Body &, Organ Weights in Male CBA Mice, Intact and at Four Days After Adrenalectomy •• 62 8. Mean Body Weights and Mean Absolute Weights ot L~rmphatic Or!!ane, Genltal Organa, and Adrenals of CBA Mice. • • • • • • • • • • • • • • • • •• 77 9. Mean Relative WeiiY,hts of Lymphatic Organs, Genital Organs, and Adrena1s of eBA Mice • • •• 78 10. Proportion of Lymph Nodes, Thymus and Spleen in fl'otal Lymphatic Organs of Normal eRA Mice • •• 96 11. Absolute and Relative Loss in Body Weight at One, Pive, and Ten Days After Adrenalectomy in Ma.le aDA lidos • • • • • • • •• • • • • it • • • ••• 113 12. Absolute and Relative Loss in Body Weight at One, Fi ve, and ':[16n Days After Adrenalectomy in Female aHA Mice • • • • • • • • • • • • • • • • • • •• 114 13. Absolute and Relative Weights of t4.ale and Female Genital Organs, Normal and B.t One, :Wive, and Ten Days After Adrenalectomy in eBA Mice • • • • •• 117 vi Table 14. Absolute and Relative '~\ieights of the Total Lymphatic Organs, Normal and at One, Five, and Page Ten Days After Adrermlectomy in C!::;a Mice • • •• 119 15. Absolute and Relative WeIghts of the Total Lymphatic Nodes, N~rmal and at One, Five, and Ten Days After Adrenalectomy in GBA Mice • • •• 125 16. Absolute and Rela ti ve Weights of tr:e PerIpheral Nodes, Uorma1 and at One, Five, and Ten Days After Adronalectomy in CBA Mice •• • • • • • • 130 17. Absolute and ftelative Weights of the Mesenterio Nodes J Normal s.nd a tOne, Pi va J ;;\nd Ten Da.ys Aftc,r Adrenalectomy in GBA Mice • • • • • • •• 135 18. Absolute and Iielati1,'s Wei.shts of the Spleen., Normal nnd at One, Five t B.nd 'ren Days After Adrenalectomy in C;3A !lica cf Var~ous Ap:es • •• 139 19. Absolute and Relative We:c:hts of' the Thymus,. Normal and at One, Flve, and Ten Days After Adrenalectomy • • • • • • • • • • • • • • • •• 146 v<1i Figure 1. LIST OF ILLUSTRATIONS ChanG:es wi th Age in Body Weight In Male and Pemale CEIA riliee • • • • • • • • • • • • • • • • 2. Normal Growth and Involution of the Inguinal Nodes • " • • • • • • • • • • • • • • • • • • • 3. Normal Growth and Involution of the Axillary }lIodee • • • • • • • • • • • • • • • • • • • • • 4. l'Jormal Grcwth and lnvolut.ion of the !'i~esenterie "tiodes • • • • • • • • • • • • • • • • • • • • • 5. Normal Growth and Involution of the Thymus • • NOrl:1al G:r.>owtu and Involution of the Spleen • • 7. Normal Growth and Involuticu of the Adrenal Glant:.s . .. . . . . . . . . .. • • • • • • • • • 8. Normal Growth and Involution of the rfestes • • 9. Loss in Body Weight of ~.lale and Female CHA hace Four Days After Ad;"onaloc tomy • • • • • • • • • 10. Alterations 1n Absolute and Relative Weights of Inguinal Nodes of '\1ale a,nd Female CBA Mice Pour Page 39 42 43 45 4'1 49 50 52 59 Days After Adrenalectomy. • .. • • • • • • .. •• 63 11. Alterations in Absolute and Relative Weights of Axillary Nodes of Male and Feme.le aLA Mice Four Days lifter Adrenalec tomy. • .. • .. .. • • • .. •• 64 12. Alterations in Absolute and Relative Weiq;hts of Mesenteric Nodes nf Male & Female aBA Mice FoUl" Days After Adrenalec tomy • • .. • .. • • .. • • ... 66 13. AlteratIons in Absolute anc' llelatlve Weights of Thy.rnus at' Male and I'ernale CT'JAMiee Four Days After AdrpnBlecto~y • • • • • • • .. • • • • •• 68 14. Al tera tiona in Absolute and :1elati va Weights of Spleens of' ],1ale and Female CBA Mice Four' Days After Adrenalectomy .. • • .. • .. • .. • • • • •• e9 IS. Chan;:;es 'Ali th Age in Body Wei~ht in Male and Fecnle CBA :.: ice • • • .. • • • .. • • • • .. • viii • • 79 • Pigure 16, 17. 13. Absolute Growth of Genital 'I'issue • • • , . .. ReIn ti ve Growth 0 f Geni tal Tissue • • • • • • :~orr1~ll Glands G.r o.w t.l~ .. a.n.. d .I n.v c.l1 ..l t.i. on. o.f. th.e .. A..d r.e n.a l . Page 81 <21 83 19. G~"owt:l and Involuti on 0 Total Lymphn tic Organs 65 20. Absclute Growth and InvoluUon of Total Nodes, Mesenteric Nodes, and Peri nheral Nodes • ... 87 21. Helati va Growth and Involution 0 r Total Nodes, Mesenteric Nodes, and Peripheral Nodes • • •• 88 22. Growth and Involution 0 f the Spleen • • .. .. • • 92 23. Growti} and InvoluLic.n of the Thymus · .. " . • • 94 24. Chan:~es wi th Age in Pro port1 em of Spleen and Thymus to Total Lymphatic 'I'issue • • .. • • •• 97 25. Crlanges wi ttl Age in Proportion of Total Nodes, Mesenteric Nodes, Bnd Peripheral Nodes to Total Lymphatic Tissue • • • • • • • • • • • • • • • 99 26. Losses in Body Weis<;ht Afte~ Adrena1ector:JY •• 115 27. Al tara tiona in .l:'\.bso1u' e Wei0;hts 0 f Total Lymph'! tic Orf:',an13 After Adrenalectomy at Var' ous A.~es in CDA Mice • • •• • • • • • • • • • • • 120 28. Increase in Re1ati va ~Neights of Total Lym­phatic Organs After Adr0:'lnalectC'rny in CBA :Mlce of Various Ages • • • • • • • • • • • • • •• 121 Incy>ease in Absolute Wel',:hts elf Total Lymph Nodes After Adrenalectomy in GSA MIce of Var-ious Afes • • • • • • • • • • • • • • • • •• 126 30. Alterations in Relative Wel~hts of' Total Lymph Nodes After Adrenalectc~r'Y in CBA Mice of Various Asee • • • • • • • • • • ........ 127. 31. Alterat:i.ons in Absolute Weights of Pe:r>:i.pheral Ngdes After Adrenalectomy in CPA Mice of V~rious Ages •••••••••••••••••• 131 32. Alte~atious in Relative Weights of Peripheral Nodes After Adrvnalectomy in CBA Mice of vfirlous Ages. • • • • • • • • • • • • • • • • .132 ix Figure Page 33. Alterations in Absolute Vieights of Mesenteric Nodes of CBA Mice of Various Ac;es After Adrena1- ect0~y • • • • • • • • • • • • • • • • • • • •• 136 34. Alterations in Relative "iei~~hts of rliesenteric Nodes of CBA M.ice of Various A~es After Adrenal-ectvwy • • • • • • • • • • • • • • • • • • • •• 137 35. Alterations in Absolute Weights of Spleens of CBA I{.ice of VarIous Ages Aftet' Adrenalectomy t' 140 36. Al teraticns in Relnti va Weights of STJleerlS .:\fter Adr';nalecto::::y in CBAMlce of Various Ages • •• 141 37. Alterati.ons in Absolute 'dei,,~hts of Thymi After AdrenalE;ctorny in GBA Mice of VarIous A:~es • •• 147 38. Al tera ti(:ms in Re1D.tlve'rv'eiGhts of Thymi After Adrf.:malectomy in CBA Llice of Various Ages • • • 148 I. INTHODUCTION Hecent developments in the fields of cytology and embry­ology have indicated that the problems of aging in the indi­vidual are fundamentally related to problems of the growth of cells and tissues. It is now recognized that growth is a part of aging; that tis8ues become old as they grow, but that cells are potentially immortal in the sense that they are capable of indefinite growth and division and that the cessation of r;rowth and termination in death is due to dis­turbances in the factors controlling ~rowth. With the development of this concept, men in several fields of science have studied growth from many different aspects and much information has been gained, but the most promising field for study of growth in the complete biolop;i­cal organism is that of endocrinology. We now know that the metabC'lism and growth of the differ­ent tissues of the body are under the control of hormones elaborated by various endocrine glands, but that the inter­relationships of the glandular secretions are very complex. The unraveling of these oo~plexities may some day lead to a solution of SOT'ie of the problems of agin~, degenera ti ve diseases, and malignant growth. The mesenchymal tissues are one of the most important from tho standpoint of the degenerative diseases and the phe­nomenon of agin:~ i tsal!. These are the tissues through wh:t.oh 1 ......... 2 metabolic exchanges take place, which help resist infection and which contain cells of multipotential properties capable of replaclng certain cells of the parenchyme as they become old and die. SO~'le of the problems of aging of' the individual are essentially the problems of the depletion of the properties of these lTIultipotentlal cells, and since many of the activities of these cells are under the control of endocrine secretions, the hormonal control of the mesenchymal tissues becomes a problem of fundamental importance. The lymphatic tissue, with its reticula-endothelial cells and lymphocytes, contains a great number of cells of rrlultlpotential propeC'ties. Thus, the problem of the hormonal control of lymphatic tissue acti­vities assumes paramount importance. In recent years a great deal of attention has been paid to the biology of the lymphatic tissue, especially as its im­portance as 8. defense mechanism of the body has become recog­nized. Th.at the lymphatic system 1.8 under hormonal control has been demonstrated, but the interrelationships between the lY;'!1phatlc system and the various endocrine glands have not, as yet, been fully elucidated. Moreover, the incomplete knowledge of the normal growth of the lymphatic tissue makes it diffi­cult to arrive at any workable hynothesis concerning these interrelationships. It is toward the solution of some of these problems the t this study was undertaken. The l)nnphatic tissue is composed of s. stroma of reticular cells and elastic and reticular fiber's, in the meshes of which ----~ ------- are scattered 1'1:'6e cells of va.rious types, but mainly large, small, and medium-sized lymphocytes. In ma.mmals this tissue forms distinct organs suoh as the lymph nodes, thymus, and tria scattered lymph nodules in the mucous membranes of the respiratory and digestive tracts. It alao forms a large part of the spleen, and is found in smaller amounts in bone marrow, the lungs and liver. Kindred (1940), Andreasen and Christensen (1949), and Andreasen and ottesen (1944) have established that the forma-tion of lymphocytes takes place in the lymphatic tissue, par­ticularly in the thymus. The 1yrnphocytes enter the blood streao by way of the lyrnphatics but their fate, once they have entered the blood stream, is still unknown. All of the f1.illctlons of the l:rmphatic tIssues and the lymphocytes have not yet been clarified. It has long been held t;ltl t the ~ lympha tic tissues play an important part in the body's dofense reactions. Virchow (1860) first formulated the theory that the IYTrph nodes acted as barriers which filtered out pus corpuscles and inanimate particles carried by the lymph. Since Virchow's day the barrier theory has remained virtually un­changed, and it has become recognized that bacteria also are arrested in the glands. Hellman (1921) and Hellman and 'White (1930) extended the defense theory by their concept of the "reaction center". These authors belleved that the l:vmohatic tissues protected ,. 6 • the body against harmful sUbsta.nces circulating in the blood 4 stream. Hellman called the germinal centers of the nodes and nedules "reaction centers" and said that these removed various agents from the blood by virtue of their retioule-endothelial cells. Among additional functions whloh:have been attributed to the lymphatic tissue are: the metabolism and transrortation of fat and protein, the storage of vitamins, the oroductlon of hormones and.sntlhormones, and the destruction of and some­t!:: 1es production of red cells. A mere detailed consideration of these functtons rr:.ay be .found in the monograph by Drinker and Yoffey (1941). A most important functll)n attributed to the lymphatic tissue has been the production of antibodies. This was demon­strated as early as 1898 by Pfeiffer and Marx. McMaster and Hudack (1935) reinvestigated the problem and showed that agglu­tinin formation can take place in lymph nodes. Since that time, antibody formation in lymph nodes ha.s been demonstrated by many ~:rnJ s ti.";:J tors, Ehrich (1929£1) .. Dougherty, Chase and 'White (1944), "Nhite and Dougherty (1945), and Harris, Grim, . ::;:c­tens and Eh.l'J.ch (1945).. 'The presence of antibodie6 :Ln tissue l~~phocytes of i~~unized anL~als was Qlso demonstrated by Dou';herty et al (1944) Hnd Harris and cO-Vlorkers (1945). However, the "preoise oe1l t:rT>e in which the firs t synthes is of im~1Une 71obuli.n occurs following an injection of antigen has not yet been demonstrated. It has long been recognized that the structure and functions 5 of the lymphatic tissue are regulated by hormones. In 1855 Addison noted. a lymphatic tissue hyperplasia in one of the eleven cases he described as "Addison's Disease". Since then an overwhelmimt, amount of experir:1Emtal and clinical evidence has accumulated to indioate a close relationship between the adrenal cortex and the lYr.lphatic tissue. In !.-lis first aocou..'1.t of the adaptation syndrome, Selye (1937) demonstrated that thymic atrophy could be mediated by pituitary and adrenal cortical stimulation. Since Selye's work the pituitary-adrenal-lymphatic tissue relationship has beco~e recognized as an important part of the body's reaction to a variety of stre(Js st:Lmuli includlnr; bacterial infections. However, the mechanism by which the adrenal co~tex regulates the ':'lOrmal fl;rowth of the l-:Tmphatic tissue is not lrnown. The studies that have been made indicate a complex interrelation­ship between the adrenal-cortical horMones, sex hormones, thy­roid and perhaps the pituitary growth hormones in the regulation of normal lymphatic tissue growth. The importance of investi­gating these relationships becomes apparent when it is seen that the treatment of CBA mice with estrogenic hormones for periods of two to ten weeks leads to certain chromosomal aberrations in lymphocytes of the spleen, thymus and lymph nodes, Santisteban, Scbneebeli, and Dougherty (1950), and that ten weeks of horrJone treatment is sufficient to induce leukemia, Gardner and D:,U'-dlerty (1944). A further argument in favor of investigatin~s these relationships is add.ed by· the recent review by DeAsua (1950), wLo is led to believe that The leukemias develop in subjects with a functional depression of the bone marrow (hypomyeloid states) whioh in some cases is acco~panied by hyperflIDctlons of the lymphatic system and that such anatomical modifications depend on a disturbed equilibrium of the endo­crine system, essentially caused by constitu­tional states of adrenal cortical insufficiency. He further adds, If such an organis~ is submitted to stimuli (infections, intoxications, emotions) that a normal ind ividua.l easi ly overcomes, condi tions would be produced in which the 'star;e of resis­tance would easily give way to a stage of' ex­haustion, and in this sense \ve can place the leukemias in the group of secondary diseases due to insufficient response of the endocrines to stress, with a consequent breakdown of the structure and funations of the hernatopoietic apparatus, constitutionally damaged b~ the primary enciocrir:ec;e::rturba tion. Then, the mesenchymal tissues, the reticulo-endothelial ays tam, free of the brake Im:)osed by the pi tui ... tary acirenal-cortical nppSr'8. tus, would start to proliferate,likely giving rise to the dis­turbances that characterize the leukemias. De As us bases his views on clinical observations, and ex')erirlental evidence has yet to be BU.bm! tted. Before such an hypothesls can be tested, a better understanding ot' the normal growth of tb.e lymphatiC t issue in a sui table experimental animal must be obtained, Bnd the relationships of the endocrine hormones to normal growth must be clarified. With such objeo-ti ves in mind, this study of the normal'~ro\Vth of lymphatic tissue and its relationship to the adrenal gland in the mouse was undortaken. 7 II. REVIEW OF LITERATURE fA. The Normal Growth of Lympha tic 'I~iBaue A review of the literature concerning studies of the role of the thymus in re lH. tion to health and disease is nresented by Boyd (1932). Studies of' the normale;rowth of' lymphatic tissue are reviewed by Andreasen (1943). In this review a.n attempt is made tij integrate the more important lc;own facts about the normal r:l'owth of lymphatic tis<ue and its rolation to the adrenal cortex. Early interest in the thymus centered around observations of enlarged thymi associated with sudden death whIch ultimately led to a still persistent belief that enlarged thymi represent a constitutional susceptibility to death from trivial causes. Death had been considered due to pressure ot the enlarged thymus on the trachea. Friedleben in 1858 denied this association and since then there has been a con­tinued discussion as to the exact role the thymus plays in sudden and unexpected doa th, Br)yd (19:32). In 1889-1890 Paltouf developed the theory about the lymphatic-chlorotic constitution, "the status thy:mico1ymphati­cus", in which the prominent lymphoid tissue and thymus found in children and in young adults dyinG' suddenly of no demon­strable cause represented an abnormal constitution in whlch the person was highly susceptible to death from trivial causes, Boyd. (1932). Hamlnar (1905) found that the thymi of healthy persons oyin:::; of accidental causes weighed as much as those 8 o.f persons dying .from "status thymico1~rmpha.ticusff. Since then, Har:Ir:'larts results for the postnatal period have been confirmed by many authors, and it has 'been established that the concept of' status thymicolympha ticus as a cO'lsti tutiona1 anomaly is based on erroneous ideas about the normal size and structure of the thymus. A few anatocl1sts in the earlier days recognized that gen­eral agencies, primarily nutritional disturbances, were able to bring about a diminution in the size of the thymus. Simon (1845) called it a "barometer of nutrition and a very delicate one". However, full credit for the concept of "acciJental invol­ution" beloIl::";s to Hammar who in 1906 substantiated t'00 role nut.ri tional deficiency plays in reducing the size of the thymus. Jonsen (1909) at the sugr:;:est1on of Hammar further investigated this question and by weighing the thymi in rabbits established that starvation produces a rapiCi and violent reduction in thymus parencflyr:1a at a ra te tha. t has no counterpart elsewhere in the organis:a, Antlreasen (1943). Thts early interest in the size of the thymus led to studies of the normal changes in the thymus with increasing age and much infor.mation has been acoumula ted both for the human and lower animals. Waldeyer as early as 1890 demonstrated that adilJose tissue infiltrates the thymus with increasinrr, age. FU.rtl-ler attention was called to thIs by Ha:rrunar (l906), who worked out a method of !':1aking an exact determina.tion of the amount of medulla, cortex and connective tissue in the thymus. 9 Jackson (1913) studied the variability of tissues during growth in the rat and found that the most variable were the thY-11mB and ti:",le spleen. Hammar (1921) determined the changes in the amounts of cortex, medulla G::1d connective tissue in the thymus of the hurnan frm the age of three months until eleven to twelve yea.rs and found a large decrease in cortex and medulla and an immense increase in fat and connective tissue. Thus, whiJ.e we know much about the normal growth of the thyrnus, there is still much to be learned about the norniB 1 growth of the other lymphatic organs. As yet, no complete studies on the growth of the entire lymphatic tissue in oman have been reported. T~u s has been a ttempted only in the rab­bit by Hellman (1914) and to a. lesser extent by Reinhardt (1943) in the rat. The difficulty underlying studies of the total lymphatic tissue 1s the wide distribution in the lymph nodes, spleen, and mucous ~embranes of the digestive tract and respiratory passages. Andreasen (1943) estImates that there are between .:Civ'J hundred and one thousand lymph nodes in man. Since the techniques for such estimates are laborious, other studies have been based upon attempts to assay the total size of lymphatic tissue from selected structures. Although data obtatned. by sampling are incol:1plete, the method allows the use of many more animals and therefore gives greater signifi­cance to the results. The broadest critical reviews concernIng studies on 10 specific lymphatic or-gans are those of Hammar (1936) on the tLymus, Hellman (1930, 1943) on the lymph nodes, and Hellman (1930) on the Belsen. A general rev~.ew of the biology of the lYI:'Ipha t~.c system is 101 ven by Drinker and Yoffey (1941, 1949). Jolly and Rossello {1909} reported that the Malpighian bocies of' the rat spleen attain definite structure at two months. They obtained data on the growth of lymph nodes in rats and rabbits but roost of the observations were not quan­titative to any extent. Scammon (1925) found that the thyrnus and lymph noC.,es have the same unIque tyr)e of growth ~urve and that ~he th:rmus has a closer relation to body weight than to length, and that it InoI'eases at a faster rate than does body weight. That the maximum weight of the thymus is attained at puberty was observed by Soderlalfd and Backman (1909) in the rabbi ts Ii by Hmn.'TIar in cartilaginous flanes (1932) and in man (1926). Boyd (1932) reviewed the work on growth of the thymus in man and confirmed Hammar's finding that the greatest w6ie;ht of the thymus is at puberty. Andreasen (1945) demon­strated that in the male guinea pig the maximum weIght of the thymus occurs in the second month, whereas in the female it occurs in the fourth month; and that tl~.roughout the first year the thymus is larger in the females. The ti:ne of attain­ment of ::1a.x1mum weight of the th:srmus in the rat has been de ... LC'''''lined by many workers.. The results indicate a wide varia­+'~') n from four days, IVatanabe (1927), to one hundred fe>urteen 11 days, Winter (1924). Chiodi (1938) found the maximum thymus wel.'~ht nt sixty days in the female and at ei~~hty days in the male. PIlE,,"G (1941) found the tn8ximum weights at sixty days in both sexes. '1':".0. t regress! va changes in the lymphatic tissues take place after attain.ment of maximum wel o;ht has been 1:':1ost readily demonstrated in the thymus. Soderland and Bachman (1909) reported on the anatomical changes in the normal thymus.with increasin;~': a--:e in rabbits. (_troll (1928) studied purine base as an lndlca tion of numbers of l'jt'TIphocytes in man and found a decrease with senescence. lImen (1926) found that the l-ymphoid tis2ue of the spleen in man increased up to sixteen to tW6::1ty years and was then followed by a decrease. Andreasen (1942.i) studied the weight changes and the changes of nuclein p'b.osphorous with a:",;e in the thymus, spleen, and representat.:tve samples of subcutaneous and InC) santeric lymph nodes in ra ts. He found that the nuclein phosphorous content of these crgans serves as a measure for the amount of lymphoid tissue. The grovi'th of lym~hoid tissue. in the thyelUs, lymph nodes and spleen was found to run parallel with the general ~:c;rowth of the animal until UIe end of the third or fourth month but that the rate of erowth dlU'lng this period varied. The growth of the thY1:iuS was retarded about the time of pubert)T (second month). At t1:lis time a retardation of growth could also be olJserved for the lymphoid tissue in sO!~e groups of lymph nodes. but not for the splenic pulp. After the end of the 12 growth perIod the lymphoid tissue underw'ent an involution which was most pronounced in the thymus and was manifested by a reduction in both absolute and relative weights. The involution of the lymph nodes with increasing ap:e dld not af-feet tlleir wei<::ht.s, but the absolute and relative amounts of lYl':1phold tissue underwant stat1.stically significant reductions. In the spleen there was a reduction in the relative amount of 1;'1;1:;h01 d ti ssue only. A study of the weight changes d'JI'ing aglng was also carried out by Reinhardt (19~3) in the rat. In general, his results agreed with those of Hellman (1914), Chioui (1936), ane: Andreasen (1943). Denz (1947) traced the histolo:J;ical cha.nges in human lymph nodes froe infancy to senility and found age differences re- 1ated to their function and anatorieal position. He described the h:lstolo(~lca1 alterat;ons in detail. Andrew (1946) and Andrew and Andrew (1948) found that age changes in the 9?leen and deep cervical nodes of one hundred Wistar rats were g-rs8t enough to permit histological identifications of nodes from youn , mlc;d.le-a.ged, and senile animals (cited by White, 1949). B. 'l'he Normal Growth of Lyr::phatic Tissue and the Adrenal Gland 1. Effect of adrenalectoIDZ Adrenalectomy has been performed by many workers to note its effect on lyr:1Phatic tis:;;ue, but in al::::1ost every C9.se only the effects on the thYlnus have been observed. 13 As early as 1899 Boinet reported an increase in the size of the thyr:ms in eleven out of .fifty-nine rats following adronaloc tomy. Similar f'indin{l;s were reported by Calogero (1924). However, both experiments were poorly controlled. Heavier thymi in cats following adrenalectomy were also re­ported by Auld (1899), Aub at a1 (1922) MacJv:ahan and Zwemer (1929) and Zwemer and MacMahan (1929). In the human, We'isel in 1899 noted B relation between diseased adrenals and en­larged thymi. Hedinger (1907) called a ttentl on to the fact that in many caSBS of status lymphaticus, diseased adrenals were also present. Diseased adrenals associated with enlarged thymi were also found by Hart (1908) and by PappenheiMer (1910). Crowe and 'Nislocki (1914) stud1ed the effects of piece­meal ablation of the adrenal in dogs and reported ~nlarged thyrai in fO~J.r out of twenty aniInnls which survi ved subtotal adrenalectm-:1Y for two weeks to six :-:1onths. However, in rab­bits unllateral adrenalector::1:T did not cause any alterations in ~~he thy!'1us, Marine Manley and Baumann (1924). They also reported that total adrenalectomy "not only delayed involution bu.t caused the regeneration of the involuted thymus". TIley based their conclusiQ),s on gross and histolo;-r,ical examinations but presented no weight data. rr'ake and Marine (l923) point out that increases 1n the weight of the thymus and lym'-:,hatic ttssue following adrenalectomy frequently occur. Jaffe (l924a) publlshed a detailed account of the effect 14 of adrenalectomy on the thymus of a.lbino ra.ts over one hundred e1.ghty days old, after the thy::lUS had already undergone a. marked involution. He observed an increase in absolute weight of tbe thymus, in contrast to a lo!~s in body weight. Thls effect was r:mximal between three and fi ve weeks following the operatlun. Jaffe (l924b) substantiated the fact that adrenalec­tomy resulted in the regeneration of the invol\lted thymus by making histological studies of the thymus before and after adrenalector.1Y. Jaffe (1924a) also indicated that l,ypertrophy of the thyrI1us after adrenalectomy was greater in the fe';'!ales. Eowever, Marine, Manley and. Bau.mann (1924) failed to ftnd a '.,' ifference lr:. the wel:.';ht of the thymus of adrenalectomi zed fe~nle end male rats or rabbits. Jaffe (1924b) recognized the i~portance of assayi the effect o!.adrenal extirpation on the thy~us against the growth rates 1.. the normal animal. His investi(~ation f~howed the.t wi thin fi V~ to six days after adrenalec tomy, thyrrd.c enlarge­r: 1ent is de tectable if the anhnal is in r~ocd condi tlon. The max:t~!lunl enlari:ement is reached three to f1 ve weeks after abla­tion and theellef is expressed that there follows a decline in weIght which <ioes ':lOt approach the normal for a period of months. It is further suggested that the t,:,yperplasia is more marked in females than in males. Juff'e (1924c) studied thymi of ":rowing rats and found that if adrenalectcrny is per­for','; 18d B t tr~:trty-fi VEl to fifty-four days of age the thymus exhibl ts addl tional ,~rowth, tile maximum effect be inr: reached 15 at two weeks after able tion. From these findings it 1'las sug­r: ested that thynic hyperplasia. after birth is due to the degen­eration of the adrenal cortex. Housssy, del Castillo and Pinto (1941) adrenalecto:~'liznd rats at sixty, one hut'1.dred, or one hundred eighty days of age and sacrificed specirnens of. each group at intervals of ten. twenty, and thirty days 1e tar. lJ.'heir resu1 ts confirmed those of other YifOrl;::ers r.nd they sugu;ested that Ii true hyperplasia of the thymus occurs after edrena lectomy .aapela (1944) also der:::onstrated a true hyperplasia of the thymus in adrenalec-tomized ra 1,8 which was maxIma I at abC'clt th.trty-days. The mS.ximal welr~hts were slightly hir.-;her in females. Heichardt ana. l.,1 oI· roes (L940) found that there was no involution of the th.ymus and that there was an increase in weight in the sys­temio 1y~ph nodes in adrenalectomized male rats maintained for fort:r ... five days on a diet to whioh 17~ sodium chloride had been added. Dougherty and Woodbury (1949) evaluated chane:es in thymus weights of adr6nalectomized mice dllring the prepuberal, puberal, and postpuberal ages with those of' nor:ma1 mice of the sa::16 age and found that at any age the absolute and relati ve wein;hts of the thymi of adrenalectomized males Vler'e greater than that of the controls. In the female the relative weight was greater but the absolute weir:;ht remained the SHme. However, on a 'we lrr.ht basis, i:"lcie;-)endent of age, the re Is tion b(cd;ween thymus wel!:;ht and body weight was indistinguishable fror1 that of the 16 cont~o1s. Since there is Q loss of body weight following adrenalectomy the maintenance of the oontrol body weight­relative thymus weight pattern implies that as the body weight deoreases the relative thymus weight inoreases. Selye (1936) suggested that the enlarged thymus following ad~enalectomy is not due to a real hype~p1asia but to the fact that various agents cannot produce 1.nvolution and thereforo the thymus is restored to its pre-involuti-.'nary state. However, a histological study by Parhone and Cahane (1937) on the enlarged thymi of rats five days arter adrena1eotomy showed increased vascularization, a better developed cortex with an increased D~~ber of lymphocytes, and in the medulla there was an increase in Hassal's corpuscles. Reinhardt and Holmes (1940) reported no specific morphological change in the thymus and lymphoid tissues of adrenalectomized rats, other than an actual increase in the bulk of elements normally present. Rapela (1944) reported a marked hypertrophy of the lobules, which was due to a greater increase in the size of the cortex than the medulla. The interlobular fatty tissue disappeared. There was no change in the size of the thymocytes, but their numbers were increased in both zones. Gregoire (1943) provoked acute involution of the thymus by X-ray and made a histological study of its regeneration. He found that there seemed to be a stimulation of the epithelial componellts but that much of the weIght increase was due to regeneration in the cortex. Since an increase in size was also 17 found in the lymph nodes, he sugf?,ests that the epithelial stimulation may be specific only in the thymus. Jaffe (19:24a) found that lonser periods of adrenalectomy in rats x'esu1 ted in a depression of the amount '.)f increase in th.e weight of the thymus. Houssay et a1 (1933) also round a ;r,reater thY1~ic weight at. thirty days after adrenalectomy than at one hundred days. Similar results were reported by Rapela (194:4) who found an atropby in the thymus at fifty days after adrenalectomy in contrast to the hypertrophy at thirteen to twenty days; this he accounts for by suggesting a hyperfunctlon of regenerated accessory adrenals which are lnade up of only cortical tissue. Jaffe (1924a) explained the effect of adrenalectomy on the thyt:1US by postulating an upset in the interrelationships be tween the gonads, adrenals and the thymus •. Marine, Manley and Baumann (19~4) speak of a similar relationship including a role pla.yed by the thyroid. However, Rapela (1944) reported that ablation of the gonads and the thyroid did not prevent 'hypertrophy of the thymus a.fter adrenalectomy. 2. _E__f. .f...e...c....t._ _o_f_ _a_d_r_e. ..n....a_ l_ e_x. ..._ trac_ts ,. a....n...~ d _a_d__r_e_n_a. ...l.. ....c....o; ;..or.... t...i....c....a....~ l . s...t..e_ _r_o. ....i...d, .;..;,s.;;;. !.!ost of the investigatIDns on the effects. of adrenal extracts I ,adrenal cortical steroids, and s.drenocorticotrophio hormones have been conducted in ~ats. Scott and Bradford (1931) faIled to find atrophy of the thymus in normal or adrenalectomized rats treated .wlth cortin. Using cortical extracts on adrenalectomized rats, Low (1938) also failed to 18 fInd chan.r:es. On the contrary I Selye (19:36) found the t purl fied cortical extracts produced thymic atro!)hy although they were less effective in the adrenalectomized than in the normal animals. Be allowed a forty-eight hour interval aft,sr the injections, but accordinz to Rapela (1944) this was too short. Carriere, ~orel Bnd Gineste t1937} and Ingle (1940) also reported atrophy of the thymus with cortical extracts. Rape1a (1944) suggests that the differences in the results reported in the literature were due to differences in the lengths of periods of the injec­tions as well as differences in dosages. ':!lells and Kendall (1940) and Kendall (1941) found that the amorphous fraotion was ~aod for the maintenance of life but did not cause atrophy of the thymus. Ingle and Mason (1938) an,j Ingle (1940) found that the oral administration of cortin (10 co/day in drinking wa.ter) produced a tro of the thymus in the intact but not a very marked atrophy in the hypophysec­tomized ant'1'1a1s. Ingle (1940), Selya (1940) 8Q d ~',elye and Albert (1942) used desoxycorticosterone and desoxycort1costerone acetate, and obtained atror .. h;,{ only when t:iven 1n l8.(>12;e doses for a oerlod of less than two months. Kendall (1941) found that dehydro­corticosterone, corticosterone acetate, 0-17 hydroxy­dehydrocorticosterone (Compound E) cause atrophy of the thymus while desoxycorticosterone, desoxycorticosterone acetate and the alrlorphous cortical fraction do not. Se1ye (1941) showed tha t desoxycortlcos te:r.'one aceta te produced a trophy :!f the 19 spleen, thymus and adrenal cortex when administered for a short term. After two months of treatment, there was enlargement of the spleen and thymus but not of the adrenals. Similar obser .. vatlons were reported by Carnes, Ragan, Ferrebee and O'Nell (1941). Selye suggests that there is a dissociated adaptation of the animal to these hormones. Thus, the effect of desoxy­corticosterone on the thymus may be lost and the atrophy of the adrenals permits enlargement of the thymus and spleen. Rapela (1944) disct~8es the capacity of "the various adrenal cortical extracts to cause atrop!1Y of the adrenal cortex and the thymus and says that the most active are corticosterone a.~d its derivatives, dehydrocorticosterone and 17 hydro-ll­dehydrocorticosterone (Compound E of Kendall). Rapela (19<~4) using female rats noted that desoxycortico­sterone acetate does not produce a si~nificant atrophy of the thymus and suggests that sex differences in responses may in part explain tho controversial results re?orted in the litera­ture. In~le (1940) observed that equal doses of cortin given to males and females resulted in less atrophy of the thymus 1n the females. Selya (1940) also oompsred the effect of similar dosages of desoxycorticosterone acetate in male and female rats and found a more marked atrophy in the males. Kendall (1941) suggested that the atrophy of the thymus produced. by a large amount of desoxycorticosterone (.100 milli­grams per day) may be due to toe .formation of progesterone or degradation products with androgenic activity. Rapela (1944) 20 points out that the various workers who have reported atrophy of the thynus usinr~ cortin or other adrenal cortical compounds ,have all used male anbmls and thus the sex of the anir.lals r:Just be taken into ac,-ou~lt when evaluating the effects of adrenal cortical extracts. Rapala, further, reported that spaying the females did not modify the action of desox,ycortico­sterone acetate but that in the adrenalectomized females its effect was greater than in the normal or in the castrates. If'hymic atror:hy was also obtaIned by desoxycorticosterone acetate in the hypophysecto:nized fC:'1ales. Repela concluded that des­oxycorticosterone acetate and probably the adrenal cortical and sex steroids have dI,reet effects on the thymus. 3. Effect of adrenoc0rticotr~phic hormone 'llhe trophic effect which extracts from the anterior hypophysis exert on the adrena.la was early reco!snized by Hous£lay (1933) .. Further isolation of this fraction enabled a more complete study of its specific effects. In addition to hypertrophy of the adrenals, 8.dminl stra tion of adrenocortico­trophic hOl'lrlOne ha.s also been found to cause loss in bod.y weight, and atrophy of the th,~us in the rat by Collip (1933), Moon (1936), Noble l~l1<l Col11p (1941), Slmnson, Li, Reinhardt and Evans (1943); and a trophy of the lymph nodes in the mouse, Dougherty and lrn! te (1943); 8!1d in the rat, SImpson, Li, Reinhardt, Evans (1940). Similar effects have been re,;-)orted in hypophysectomized rats by ColliiJ, Anderson, Thomson (1933); Davidson (1937), Moon (1':136), Evans, Moon J Simpson and Lyons (1938), ereds and 21 Moon (1940), Simpson, Ll, Reinhardt and Evans (1943) f Simpson Evans and Li (1943); and 1n castrated animals by Davidson and !,~oon (1936), Davidson (1937), Moon (1937a, b), Grede and r;:oon (1940). On the contrary, atro~)hJ' of the -thymus was not obtained in the adrena.1ectomlzed animals by Davidson (1937); Grede and t,:oon (L140) nor in the adrenalectomized castrated B.ntmals by Davidson (1937). Emery and Winter (1934) adminis­tered tIle anter'ior pi tui tary to t'-,yro:Ldecto,:dzed animals and failed t. get a hypertrophy 0.· the adrenals. However, Houssay at a1 (1933) studied the effect of an extract of the anterJor pituitary and reported hypertrophy of the adrenal even in the absence of the thyroid (quoted from Rapela, 1944). From the results thus far presented, it may be assun:Jed that the atrophy of the tbymus following .the administration of adrenocorticotrophic hormo11e is d,;"6 tc the production of adrenal cortical steroids; this hypothesis is furth'3r sup;)orted by the work of aape1a (1944), who gtudlad the effects of adrenocorticotrophic hormone in normal and Bdrenalect~~ized rats. He obtained atrophy of the thymus in the whole but not in the adreDalecto~!zed animals. Rapela further disousses the lmportance cf relating dos­a.~ e to body weight (1944). He ;)oints out that most workers us:in.'~ rats of three to four w,,~ek3 of age have obta.ined a relntiGuship between the size of the dosage and the amount of atrophy; accordinG to ti:oon (194.0) rats of four days of age showed a .?;rea tel" thymus a trophy and adrena 1 hyper'trophy with 22 with lower dosages of adrenoeorticotroph1.a hormone than did rats of twenty~one days of age. Rapels used rats of ninety-eight days of age and found no difterence between the weights of the adrenals of intact ;!);limals treated with high and low doses of adrenocortlco­tro:. hic hormone, whereas the th:::rmus showed an a trOi)hy wbt eh was proportional to the dose. On the other hand, with intact rats of twenty-one days of age, Mendive ( ) found atrophy of the thycIlUS but the hypertrophy of the adrenal was not marked. Rapels sug:;:ests tha t hypertrophy of the adrenals followlng adm1nistraticn of adrenocorticotroptd.c horMone is Dore marked 1n YOUXlP: animals because of a body weight ... dosa~e relationship ... Emery and Winter (1934) failed to find hypertrophy of the t!.-:ymus in rn ts of les s tha.n th5.r t:T 6.ays of age; hmvover, the hoI' ones used were ~:ot put'e and the dosap:es were .Low. ~apela {1944} concl~des that it is the cortical tifsue W;';iC:l C'xertE a ;"']octerating effoct on the develo)U1Emt of the thy-mus because: 1. Acirenalin produces no atrophy of the thymus in adrenBlecto~ized animals. 2. "rhe rege.nerated cortical tissue of the accessory adrenals produces atrophy of the thymus. 3. Si~nilar effects may be obtained by administering adronal cortical steroids or adrenal cortical extracts. 4. Adrenocorticotrophic hOrl'10ne s cause atro";hy of the thy;:us 1n CBS tra. ted, 'hypoDhysectonizod I but not 23 1n adrenalectomized animals. 5. A trophy of th.s tt'Jnnus may l)e obta lned in aurenalec­tomlzed animals by parabIosis with non-adrenalectomized from the adrenalec tO~j '[ zed paI'sblont. 24 III. EXPOSITION A. Scope and Purpose of Study A review of the literature has yielded an abundance of information on the normal growth of the thymus in the rat, the rabbit, and the h~an, but information on the normal growth of other lYl':1phatic organs is lacking. Similarly, much has been done to elucidate the endocrine interrelationships which help control the normal growth of the th.ymus in the rat. "However, only a few authors have given any co~.sideration to hormonal controls of the normal pattern of growth of the other lymphatic organs. It was therefore proposed to mak::e a study of normal body growth and the normal growth 01' various l~rphatic and non­lymphatic organs in eBA mice to (.ctermine: (I) The changes w:bich occur in body weL:hts of male and female r.1ice. (2) The chanzes in absolute weights of the lymph nodes, thymi and spleens. (3) The changes in absolute weights of the adrenal glands, kid­neys, hearts and livers of both sexes and of the testes of males. (4) The rel8. tionships between the weights of the various organs aud the body wetghts. This was expressed as milligrams of organ per one hundred grams of body weight. (5) To determine the sex differences in body growth and in the growth of the various organs. From theae data it was hoped to establish normal growth curves which could be used as controls in experiments involving conditions which alter body and organ weights. It was also 25 hoped to obtain information which would be useful in studies designed to elucidate the relaticnshlps between the growth and involution of the lymphatic organs and the changes in the endo­crine functions of the adrenal glends, testes, and pituitary gland. It was further proposed to induce experi7rlental adrenal cortical insufficiencies by the ext:trpation of the adrenal glands in mice of the same ages as those used in the studies of normal '~rowth, and to obs{~rve the changes in body weights and in lymphatic organ we.! ghts 9. t various pericds of time after the adrenalectomies. By cOt'lparing the normal organ wei~hts at any age with the wetp;hts after adrenalectomy at si~ilar ages, it was hoped to determine the role of the adrenal cortical secretions in controlling the growth of lymphatiC tissue and the chan;;:;ss whicr"; take place in teis relaticnship during aging. Also, by compaI'ing the weight a 1 tera tiona in the organs of the male with those or the remales :t.t was hoped to determine whether or not sex influenoes the adrenal cortex­lymphatic tissue relatic;nship. If these relationships can be established. it ma.y be possible to assay functions of the se­cretions of the adre:nal cortex by obtaining the changes in weights of the lym9hatio organs Etnd it may lead toward the control of certain diseases characterized by the overgrowth of lyr:rphatic tIssue. It ,.: ... ':r,ht thus be sUI!1marized that the maJor aims of these ex!)er1:~ents were to determine the responses of dif'ferent 26 lymphatic organs to the nermal aging process and to adrenalec­tomy at successive ages. The results which are to be presented Vlere obtained from four experi:"lents. First, a study ws.s made of general body growth and of' the growth 0 f lymphatic organs, gona.ds, a.nd adrenals from birtb through puberty and sexual maturity. Second, a study was made of the changes in body weight and changes in the weights of lyrrDhatic and non-lympha tic organs four days after adrenalectomy in animals ranging in a~e from three througb twal va weeks, 1-.!. I prepubertal, pubertal, and early sexually mature periods. ThiI'd, a study was made of the normal p;rowth and involution of lymphatic tissues from ,the pre­pubertal period to senility_ P:.Hlrth, a study waslyacle of the changes in weiqhts of the body, lymphatic and genital tissues a tone, fi ve" and ten days after adrenalec tomy in "nh--nr:l Is of prepubertal, pubertal, sexually r.'lBture, and sen:Lle ages. D. Materials al1d General Methods The anil::als used in this series of experinents werf:1 Y:1ale and virgin fet~ale mice 0 f' the CBA strain. Since the thymus 1s Be susceptible to changes in the environr:1ental co dltions, special attention was given to trle care of the animals. They were bred and kept in wooden boxes which had a sheet metal floor ana rE;lTiovable tops made of hardware cloth. Feeding bins of hard-ware cloth were built into the tops and a constant vigilance was maintained to keep the food bins adequately filled. The diet consisted of "Purina. Dog Chowlt checkers. F'resh water was 27 available at all times. The animal boxes were cleaned at least every two weeks, at w::lioh times pregnanoies were recorded, the ages of the new li tters wen"e detertlined. and old 11 tters were weaned. The age at weanin!7, was about twenty-eight clays; at this time the sexes were separated and ani'rJals of the sar16 age were rylaccd in the same boxes regardless of parentage; thus the results presented are fr::,:r.:l random samDlos of the colony rather than from spec ific litters. The animals were housed in a room kept at a ta:n!Jerature cf 80 degrees, plus or minus two degrees. It was not possible to centrol noise a.nd lightir,g which, at least in so:ne of the ex-periments, seemed to alter the results. Adrcne.lecto{;'lY wns performed via the dorsal approach under ether ane~t1,esia.. Prior tu autopsy the total leukocyte and dLfferential blood count W'c.s made on blood samples from the tail vein. The animals were sacrificed by q'.lickly pulling tiLeir heads I thus severing the cord, or by plse lng them in a jar containin,,; cotton saturated with ether. At autopsy the body weight was deteroined to the nearest one-tenth of a gram on a triple-beam balance and organ weights were determined on a torsion balance, accurate to one ... tenth of a milligr,!:'m.The arithr:16tic x:Jeans of the absolute body weight and of the absolute and relative wei~lts of the organs were determined. standard deviations were calculated according to the formula V £cI~ (#-1 ) mula and standard errors accordlng to the for- • 28 c. Discussion and Definition of Terms 1. Growth and involution Growth is one of the l:lOst cb.aracteristic pro!)erties of llvln~ catter artd in tills sonse it becomes strictly a biologi­c 8.1 terl:h As applied in the bi logical sc iances, the term f,,:rowth ultimately refers tu the buLlding up of new protoplasm in contrast to mero increases in size, as ror example the swelling of cells when they are placed In hypotonic solutions. Growth thus refers to the metabelic activities of the living cell which result in the chemical synthesis and assimilAtion of protoplasm. In the process of r.;rowth., accessory substances are also synthesized, and thesl;:, are later elaborated into the charactel~lst:!.c structures of cells and organi.sms. Wi th the deve lopl11ent of the sc ience of embryology, the term "diff'erentiation" has come to refer to chan':~es in struc­t1. lI'e, form or function. Thus growth and differentiation are considered two separate processes which may proceed simul­taneously. Since the changes characteristic of differentiation were rl:)t observed in thls study, that term has not been used. In the biological se iences., the phenomenon of grow'th may be measured by a wide variety 0 f r:!ethcds. By tee clini cian it may be mOf.lBUrea by noting the increa8e in size and we1~ht of' the Lidi vidu.al; by the biochemj s t, it ';'lay be rleasured by de­termining the increase in the number of orio;anisms in a culture; and the hIstologist may measure it by deterr.:lintng the relative 29 number of dividing cells and mitotic figures in a tissue. The bioloJ2;ist is well aware that although growth is fun­damentally a property of youn~ organisms, a continuation of t.e growth phenomenon is also characteristic of certain tis­sues of ma ture organisms, 1.-,2,-, the continued. act i vi ty of hematopoeitic tissues, growth of hair, nails, and skin, and the periodic growth 0 f the endometrl urn of the uterus. Growth in such tissues 18 senerally balanced by a continued utIlization or destruction of its end 'ol'."oducts and in such cases weIght is but a poor criterion of the dynam:i.C nrocesses taking . place. It has already been lndicated that a certain amount of Growth takes ':)18ce 1n 1yrl'lphntic tts~~ues at all ages, resulting in the 2roducticn of lymphocytes which then enter the blood stream. However, the work of Doughertya.nd D8u,qherty (1950) sl:lOwed the. t in the 1ympha tic organs, weight ohangs'f3 may be used as valid criteria of their growth,and in this stud,. growth has been identified and mea:surcd by the increase in organ weight. Tb:ts increase nay be slow or rapid, and may occur during a sbort or a long period of t Lrne. "Involution" is another term whi ch ha,s several rneaninp;s. It may refer to the return to normal size ·after enlargement, as in thecaee of the> uterus after parturttion; to retrograde changes or degenerations; to 8 decrease in certain physiologi­cal functIons, as in the ovary at meno98use; or to the shrink:tng or shriveling of an organ. '1':'16 organs of the lymnhatic system undergo a normal, prohressive decrease in welght with ar,r,e and it Is to this proaess that tne term "involution" is applied. 30 :rhUB, a study of the weight changes with age of the lymphatic or.(~ans over the whole life span becomes a study of their ;~rowtb and involut ion. 2. Absolute and relative we:l~~hts • The weight of an organ may be expressed as its absolute cr actual weic;ht in tet"':::lS of grams or fractions of grams, or it rnay be exnressed in terms of proportion of a un! t of body we:t?:ht and des:lgnated as relative weight. In th:~ s study both absolute welc:hts and relative weights were deter:nined. The relative weig,hts wore obtained by expressing the absolute wel:~hts per one hundred grams body weight. Thi s :nakes possible a direct co,':parison of the weights of organs of 61fferent sized anlmala. It :'Jay thus be seen that the chanq;es in absclute weL.jht reflect absolute growth or i!1volutlon and changes in relative we1.,....,ht reflect relative growth or involutton •. The study of chAnrr.es in relative wei?:hts becomes important 'linen it 1s necessary to know if cbanf!eS in absolute w6:l?;ht of a.n cr::,:an are t'erely follmv;ing ehangcs L! overall body weif!.ht; furthermore, a study of th ~ s ty-oe clearly brin,,:s out differences in rates of chan'se between absolute weight of an organ and the overall body weight. 3. L,}'U1:)hatic system, lEphoid and lyrHPhatic tissue The lynrohatlc syste'm is made up of' lym?hatlc vessels and lyrnpnatlc organs. The lymol·\,9 tic vessels begin as blindly endin,:<;, tbirl-walled ulY1:1phatlc capillaries" whlch ferm dense :31 networks in most of the tissues of the body_ The lymphatic vessels '.mite and. form larger vessels, the largest of which cnpty ielto veins. '1'he l:~rITlpbatic orl?ans are collections of l;ynn11o. tIc tissue which are closely C(FlDected wi t1:1 the lymptiB. tic vessels. rrhe lymphs tic tissue, whether organlzed to form spst: ific organs or unorganized as in subepi thelial 1 'J'1llprl a tic tissue or in the bone marrow, consists of a stroma of retioular fibers and reticular cells in the meshes of whIch are scattered the lymphocytes. In early works this tissue was referred to as "lymphoid tissue". Accordl:1g: to Drinker and Yoffey (1941), Ehrich (1929a) dist1nguishes three groups of lymphatic t!.ssue: 1. Lyt:i:c>hatlc tl"'sue in the lY''!'l,')h nodes whIch has afferent and efferent lymphatic vessels, and is 13t tuateci. :tn the ly"mph stream. 2. LymphatiC tissue in the mucous membrane, wh1ch has only efferent lymDb. vessels, anG. 5s situated in the fluid streams going from ti.Je mucous Membrane into the interior of the oresnlsm. 3. Lymphatic tissue in thFl spleen which has noither afferent nor efferent lymph vessels dnd is situated in the blood stream. Ehrioh also discrirninatf~s between lymphatiC and lymphoid tissue. He co·,siders lymphatio tist'"uea as those which possess germinal centers; lymphoid tissues as those which do not. Dr-inker a:ld Yoffey considered th:~s distinction superfluous, "since the gerr:1inal center Is a structure subject to considerable variation, and may develop de novo anywhere in lymphoid tissue." They also added a fourth group of lymphatIc tissue, that of the bODe marrow, to EhrIch's classification, but reta1.ned the term lymphoid rather than lymuha ti c. 32 In recent years there has been shown a preference for the term lymphatic. This 1s a.n outcome of the recol?:!11tion of the fact that all of the lymphatic tissue Is basically the saMe in stru.cture and function, re'1ardless of its location in the body. Therefore, In this study t'1e term lym'::Jha tic tissue 1. s used to d signrite t'::U3 spleen, thymus and mesenteric, axillary, cervical and inguinal lYiC1t")h nocss. The latter three groups of' lymph nodes, being subcutaneousl~r located, are also referred to as periy.)heral or subcutaneous lymr)h nodes. Total lymph nodssr'efers to all the ly:rrv)h Dodos sarrr:>led. 33 IV. RESULTS OF EXPIERIMENTS A. The Normal Body Growth and the Growth of Lymphatic and Nen-lymphatic Organs from Two. to. Twenty-two Weeks of Age 1. Procedure Ten to thirteen male and virgin temale mice were sacrificed at weekly intervals beginning the second week after birth and ex­tending to the twelfth week a fter birth. A similar series were sacrificed at bi-weekly intervals beginning the fourteenth week after birth and extending to the twenty-second week. At autopsy the animals were weighed, and the following organs were removed, cleaned, weighed, and fixed fcr histelogical study: the inguinal ncdes, axillary nodes, mesenteric nodes, spleens and thyml. Also, the weights of non-lymphatic organs, such 8S the ac~r'enal glanos, testes, kid!leys, livers and hearts were determined~ ~lle weight of the intact animal. before death, was deb.lrmined to the nearest 0.1 gram and the weIght of the organs determined to the nearest 0.1 milligram. 2. Results Statis,tical summaries of the changes with age in abselute weights in male and female :mice are presented in Tables land 2. The changes in relative weights are presented in Tables 3 and 4. An examination of the data 1'or the absolute wei[ihts shows that as the ani .a18 aged and gained in body weight, there tended to be a de.finite proD;ressive increase in the mean absolute we1.ghts of all the organs except the thymus. The 34 Table I Normal Age Changes in Body Weights and Absolut.e Weights of Lymphatio and Non-Lymphatic Organs of Male CBA Mioe Weights of G1IiphaUo organs Weight. of Non-LimphatiO Organa Age 1Ioc17 in !!e.~ in Mm-I in Wt. in Ing. Ax. il ... Spleens Adrenal Teate. .I1clna7a Lart. Liver. Wka. Grama Node. Nodea Nodea G~tiia 2 6.1 1.2 1.2 4.0 40.0 11.6 1.1 18.1 10.1 36.5 210.0 .:t.29 ,;!;.OOH .:t. 001 .:t.32 .:t2•83 .:t1.55 .:t.008 .:t1•21 .:t4.56 .:t1.55 .:t9.5 3 1.6 2.3 2.3 8.6 45.H 29.3 2.2 31.0 105.0 53.0 340.0 .:t.43 .:t.45 .:t.31 .:t1•14 .:t2.4 .:t2.4 .:t. 28 ,;!;1.61 .,t6.88 .:t2•65 .:t<!9.4 4 13.1 3.1 2.8 19.2 49.1 41.9 2.3 14.0 193.0 61.0 766.0 .:t.44 .:t.31 .:t. 21 .:t.61 ;t3.5 .:t2.9 .:t.19 .;t5.67 .;t9.2 .:t1.5 .;t52.1 5 14.6 3.6 3.4 21.2 50.4 55.9 2.9 99.0 222.0 74.0 818.0 .:t.36 .:t.41 .:t.43 .:t.83 .:t2.9 .:t7.2 .:t.04 .;t.3.3 .:t12.99 .:t2.1H ,;!;29.9 6 17.0 5.1 4.3 31.2 51.6 79.2 3.1 103.0 261.0 86.0 955.0 .;t5.25 .:t.38 .:t.31 .:t1.7 .:t4.5 .,t6.7 .:t.05 .:t2•87 .:t14.92 .:t2•09 .;t.35.1 7 18.6 5.0 4.0 34.7 42.1 50.7 2.9 121.0 311.0 95.0 1086.0 .:t.77 .:t.34 .:t.42 .:t2•83 ;t3.22 .:t2•63 .:t.04 .;tl. 99' .:t13.63 .:t4.13 .,t64.1 8 21.6 5.6 4.9 35.H 53.1 80.2 3.7 147.0 361.0 102.0 1284.0 .:t.03 .:t.53 .:t.47 .:t2•08 .:t2.30 .,t6.65 .:t.16 .:t4.66 .:t12 .35 ;t3.22 .;t45.9 9 21.0 4.5 3.9 32.9 42.7 12.1 3.H 132.0 375.0 101.0 1192.0 .:t.31 .:t.06 .:t2•08 .:t2.38 .:t4•09 .:t.01 .;t.3.39 .:t11 .43 ;t3.12 .;t.39.4 10 16.5 5.4 4.2 25.3 40.2 ' 57.8 3.2 118.6 248.0 86.1 986.0 .:t.64 .:t.34 .:t. 21 .:t1.41 .:t1.96 .;t8.78 .:t.016 .,t6.47 .:t14.54 ;t3.75 ;t35.3 11 21.0 3.7 3.1 33.8 39.2 51.5 3.6 134.0 303.0 94.0 1112.0 .:t.75 .:t.31 .1.25 .:t1.51 .:t1.39 .;t.3.31 .:t.037 .,t6.14 .:t14.5 ;t3.33 ;t36.4 12 23.3 4.4 3.s' 30.7 44.5 61.9 3.4 138.0 347.0 104.0 1113.0 .:t.59 .:t.47 .:t.29 .:t2•29 .:t2•16 .:t7.64 .:t.14 .;t.3.12 10.5 1.53 37.7 14 23.7 5.8 5.1 27.6 34.6 68.2 3.3 • • • • .:t.31 .:t.39 .:t2.37 .:t2•03 .;t5.17 .:t.25 16 24.2 7.0 6.0 25.0 29.6 84.3 3.1 140.0 339.0 107.0 1222.0 .:t.82 .:t.45 .:t.36 .:t2.51 .:t1.39 .:t12 •23 .:t.2O .:t2.31 .:t16 •16 .,t6.08 .;t51.8 IH 25.7 5.9 5.0 27.3 32.1 18.0 3.6 151.0 396.0 115.0 1441.0 .:t.54 .:t .63 .:t.54 ;t3.H ;tl.5 ;t3.8 .:t.14 ;t3 ·55 14.28 6.06 32.3 20 26.7 5.0 4.7 24.6 29.7 64.3 3.4 143.0 396.0 116.0 1375.0 ;tl.3 .:t.32 .:t. 26 .:t3. 19 .:t1.5 .;t2.25 .:t. 19 .;t5.08 ;t27.92 .:t8•01 .:t251.H 22 21.1 4.9 5.0 20.8 31.5 90.8 3.3 152.0 411.0 118.0 1015.0 ;tl.13 ;t.32 ;t.21 .:t1.3 .:t.86 .:t11 •2 .:t.15 ;t3.29 ;t21.61 .:t7.48 .;t88.2 * Weights not obtained 35 Table 2 Normal Age Changes in Body Weights and Absolute Weights or Lymphatic and Non-Lymphatic Organs or Female CBA Mice '. Weisht. of ~~hatic Orean. Weight. ot Non-Lrmpb&tio Organ. Age Bod7 in !!e •• in ~I in Wt. in lng. Ax. ».e •• Tbiii1 ·Sp1een. Adrenal Iidn.7~.art. Liv.r. . 'Rlcs. Or_. Node. Bodes lod •• Gland. 2 6.1 .9 1.2 3.2 46.7 19.1:1 .9 69.5 34.9 191.0 .:t. 25 .:t.05 .:t. 113 .:t.32 .:t2•6 .:t1.3S .:t. 104 .:t4.08 .:t1.45 .:t1O.45 3 7.8 2.9 2.5 9.3 47.4 30.1 2.3 107.0 54.0 364.0 .:t. 29. .:t.633 .:t.24 .:t.IH .:t2.43 .:t2•17 .:t.246 .:t4.35 .:t1•84 .:t19.35 4 11.3 1.9 2.1 22.8 54.2 37.2 2.4 163.0 62.0 648.0 .:t.167 .:t. 21 .:t.168 .:t2•61 .:!:,3.1 .:t1.97 .:t.13 .:!:,3.88 .:t1.30 .:t23.66 5 13.7 4.6 4.0 2).6 61.8 53.3 3.1 188.0 70.0 797.0 .:t.48 .:t.33 .:t.39 .:t1•27 .:t2.72 .:t4.39 .:t.17 ,;t6.45 .:t1•61 .:t24.71 6 15.6 3.7 3.0 28.1:1 61:1.7 59.9 3.8 223.0 76.0 848.0 .:t.64 .:t.30 .:t.303 .:t2•0 .:t2.97 .:t4·68 .:t.44 .:t14.96 .:!:,3.49 .:t24.9 7 16.1 5.2 4.2 31.4 54.7 48.9 3.7 225.0 82.0 956.0 .:t.58 .:t.599 .:t.604.:t2.39 .:!:,3.86 .:t4.02 .:t.251 .:t1O•2 .:!:,3.72 .:!:,39.9 8 18.6 3.4 3·5 34.3 57.0 52.1 4.2 247.0 1:S4.0 994.0 .:t.OS .:t.29 .:t.36 .:t2.55 .:t3.39 .:t1•67 .:t.18 ;t5.10 .:t2•22 .:t2O.93 9 17.1:1 4.7 3.9 33.7 57.5 61:1.0 4.0' 242.0 85.0 1192.0 .:t.39 .:t.24 .:t.34 .:t1.98 .:t2•69 .:!:,3.09 .:t.261 .:t6•82 .:!:,3.19 .:!:,36.32 10 18.0 4.7 4.3 28.5 53.5 63.4 4.1 227'~0 Sl.0 996.0 .:t.36 .:t.48 .:t.45 .:t2•62 .:t2.59 .:!:.5.01 .:t.24 .:!:.5.25 .:t2•1O .:t44.44 11 17.6 4.3 3.7 31.1 54.4 51.1 4.4 212.0 86.0 801:1.0 .:t.39 .:t.317 .:t.277 .:t3.14 .:t1.94 .:!:,3.06 .:t.326 ,;t6.93 .:t1.96 .:t29.6 12 19.1 4.0 3.3 25.0 50.1 ,4.7 5.1 237.0 85.0 967.0 .:t.n .:t.244 .:t.2O .:t1.36 .:t1•27 .:t2.76 .:t.30 ,;t6.15 .:t2•26 .:t27.05 14 20.1 6.5 6.1 27.4 40.1 72.7 4.7 • • • .:t1•1O .:t.835 .:t.644.:!:,3.41 .:!:,3.94 .:!:,3.90 .:t.404 16 20.4 5.2 4.7 27.3 37.0 59.2 4.2 231:1.0 H6.0 1046.0 .:t.699 .:t.47 .:t.45 .:t2•82 .:t2.,1 .:!:,3.35 .:t.332 .:t1O•87 .:!:,3.93 .:t37.12 18 20.1 4.7 4.4 21.4 36.1 69.0 4.8 249.0 81:1.0 1134.0 .:t.925 .:t.35 .:t.43 .:t2.37 .:t1.78 .:t3.48 .:t.35 .:t13.88 .:t4.1:16 ,;t60.45 20 20.6 4.5 4.2 20.1 32.3 63.0 5.0 . 256.0 92.0 1012.0 .:t.746 .:t.445 .:t.358 .:tl •Sl .:t1•63 .:t3.75 .:t.347 .:t12 .51 .:!:.4.11 .:t60.41 22 21.6 4.5 4.5 28.3 32.93 71.29 5.8 277.0 93.6 11:21.0 .:t.7OS .:t.5 .:t.554 .:t2•1 .:t1.56 .:t4.53 .:t.257 .:t6•8O .:t.898 .±98.3 • Weight. not obtained 36 thymus reached a max~r.:ttL'TI weight at eight weeks. and under'llent a pro?,ressive loss throughout the rest of the period studied. The inguina.l and axillary nodes grew ra9id1y for fi ve to six weeks and for the rest of the period their absolute we:tghts fluctuated above and below the max:!.r:n.un weights attained. rrhe weight of the mesenteric nodes increased for eight vlee~:s and then::;raduall.y declined. Thus, significant differences between the absolute weights a.t eight "'feeks a.nd those at twenty and twent;r-two weeks were observed. The patterrs of growth of t~e spleens, adrenals, kidneys, hearts, livers and testes were similar to that of the body as a whole, i.e., the rapid growth characteristic of the early stages 0 f life was followed by e. gradual decrease in growth as the animal beoame older. An examination of the data on relative weights (Tables :3 and 4) discloses that there were T)ro'\ressive loases l.n the relative we:t::;hts of the thymi of msles and fer:lsles throughout the period studied, whereas the r:"lative weights elf the inguinal and axillary nodes increased betwEHm the second and third weeks of age. After this, their relative weights fluctuated above and below the wfde;ht attained at three weeks but there hegan to be sir;nlficant decreases in relative wel{~hts after the twentieth week of as;e. The rela ti va weights of the me sen torie :lodes increased up to seven weeks of age in the males and nine weeks in the females. After these ages were attained, the relative weights of these nodes decreased progressively with age. The adrenala 0 f male an.:t:'lsls progress! vely lost in relative 37 Table :3 Normal Age Changes in Relative Weights of Lymphatic & Non-Lymphatic Organs of Male CBA Mice W~ighta ot ~Phatic Organa Weights ot Non-Lymphatic Organa Age in ~a.LI00 Oms. Bo~ Wt! in ~s.Ll00 Gms. Bo~ Wt. in IIlB· Ax. »es. Thymi Spleens Adrenal Testes Kidneya Hearts Livers Wks. Nodes Nodes Nodes Glands 2 20.0 21.0 69.0 655.0 294.0 18.6 301.0 1149.0 619.0 3460.0 .:t1.35 .:t1•8 .:t6.9 .:t19.6 .:t17.3 .:t.86 .:t12 .1 .:t28 .5 .:t19.4 .:t93.4 3 2H.8 30.0 110.0 620.u 384.0 2~.5 485.0 1522.0 100.0 444U.0 !).35 .:t 2.25 .:t ~.7 .:t40.0 .:tl~.6 .;!;.l.9 .:t11.9 .:t95.1 .:t16 •6 .:t148.9 4 23.6 21.~ 141.0 313.0 364.0 11.4 598.0 1460.0 513.0 5:J84.0 .:t2•8 .:t1•07 !fo.01 ;t22.ts .:tIS. 9 .14.49 .:t29.6 .:!:.31.5 .:t14.99 .128 •6 5 '24.~ 23.5 146.3 348.0 391.0 20.3 683.0 1511.0 508.0 6025.0 .:t2•61 !).02 .:t1.05.:t12.98 .:t45.1 .:t.911 .:t26.9 .:!:,50.1 .:t1.32 .:t101.5 , 30.3 25.6 183.5 309.0 469.0 22.0 605.0 1539.0 508.0 5662.0 .12•24 .12•05 .:t1.4 !)0.16 .:t43.1 .:t1.3 .:t19.45 .:t71~5 .:t16 •2 .:t244. 2 7 21.4 22.1 190.0 225.0 271.0 15.8 653.0 1615.0 511.0 5822.0 .:t2•02 .:t2.7 .:t11.3 .:t16.9 .:t15.6 .1.61 .:t2O.4 .:t40.7 .:t13.6 .:t199.4 8 26.0 22.9 165.0 249.0 312.0 11.0 615.0 1618.0 414.0 6154.0 .:t2.45 .:t2•26 .11.18 .110•0 !)0.3 .1.92 .113.9 .147.1 .113.0 .1131.1 9 21.0 18.0 156.0 211.0 338.0 18.0 628.0 1141.0 480.0 5566.0 .11.95 .1.89.112.66 .117.5 .124.2 .11•22 .122.4 .142.3 .17.8 .1134.3 10 33.0 26.0 156.0 248.0 355.0 20.0 118.0 1494.0 524.0 5991.0 .11•88 .11•65 .:t9.5 .112.9 .:t,55.2 .1.14 .:t19.5 .:t43.8 .:t15.1 .:t78.8 11 11.1 14.9 162.0 184.0 246.0 17.0 638.0 1567.0 450.0 5356.0 .:t1.33 .:t1•1 !fo.8 .:t6 .1 .:t13.8 .:t,5.3 .:t14.0 .:t27.5 .:t8.4 .:t90.5 12 20.3 17.2 131.0 1~9.0 262.0 14.5 585.0 1467.0 441.0 4704.0 .:t1•85 .:t1.12 .:t1O.9 .19.65 ;t33.2 .:t.69 .:t21 •8 .;!;.l8.5 .:t1O•2 .:t16O•0 14 25.5 22.2 116.0 141.0 288.0 13.9 * * * * .:t2.32 .:t2•08 .:t7.13.:t 5.41 .:t18•24 .:t.51 16 29.1 25.2 101.0 124.0 331.0 12.8 586.0 1398.0 440.0 5041.0 .:t2•16 .:t1•83 .:t1.11 !fo.32 .;!;.l6.96 .±,.633 .±,19.01 !)1.67 .:t15.94 .:t234.1 18 23.1 20.0 103.9 127.0 306.0 14.1 592.0 1538.0 446.0 5661.0 .:t2•27 .:t2.38 .:t12•66 .±,B. 26 .±,11,85 .±,.125 .±,15.61 .±,36•21 .±,19.1 .±,151.3 20 18.9 17.6 91.1 112.0 244.0 12.8 541.0 1465.0 430.0 5124.0 .:t1•16 .:t.91 .:t1O.41 .:t6.41 .:t9.22 .:t.118 .:t 26.80 .:t40.91 .:t12.45 .:t15U.46 22 18.4 18.8 71.3 119.0 326.0 12.4 511.0 1514.0 433.0 3896.0 .±,1.26 .±,.961 .:t4.61 .:!:,5.83 .:t33 •1O .±,.59 .±,22.45 .:t29.1 .:t12.46 .:t165. 83 • Weights not obtained 38 Table 4 Normal Age Changes in Relative Weights of Lymphatio and Non-Lymphatic Organs of Female CBA Mice Weights ot ~phatic Organs Weights ot jon-~phatic organs Age. in Mlmia.l100 Gas. Bo2;[ Wt. in line .L100 Qu. Bo2;[ \ft. in Ing. Ax. )(es. TbylI1 Spleens Adrenal Kidneys Hearts Livers Wk •• Node. Nodes Node. Glands 2 16.0 20.0 52.0 713.0 311.0 15.1· 1133.0 574.0 3115.0 .±1.08 ,;t3.06 .±5.39 .±17.3 .±12.39 .± 1.19 .±22.05 .±9.37 ,:t56.4 3 35.2 31.2 118.0 613.0 412.0 29.0 1380.0' 698.0 4617.0 .±6.77 .±2.14 jfJ.78 .±25.79 .±31•O .±2.63 .±23.5 .±17.48 .±97.7 4 17.5 18.3 212.0 473.0 328.0 21.2 1440.0 547.0 5731.0 .±2.09 .±1.52 .±25.4 .±25.'(6 .:!:.15.76 .:!:..95 .:!:.21.78 .±9.71 .:!:.225.5 5 33.5 30.3 172.0 449.0 424.0 22.4 1373.0 513.0 5827.0 .:l;.3.12 . .:!:.3.38 ~.16 .:!:.19.28 .:!:.47.1 .:!:.1.09 .:!:.46.4 .±9.63 .:!:.159.5 6 23.6 19.3 183.0 446.0 380.0 26.2 1415.0 488.0 5488.0 .:!:.1.56 .:!:.1.6 .:!:.10.03 .:!:.26.0 .:!:.20.05 .:!:.2.22 .:!:.78.2 .:!:.14. 02 .:!:.180.7 7 31.8 26.0 195.0 342.0 301.0 23.0 1414.0 510.0 5934.0 .:l;.3.5 .:l;.3.44 .:!:.9.96 .±25.9 .±17.41 .±1.14 .±53.3 .:!:.13.4 .±91.7 8 17.7 18.5 184.0 308.0 292.0 21.5 1323.0 452.0 5359.0 .±2.8ts .:!:1.76 .:!:13. 22 .:!:16.tl ;t8.67 .:!:..89 .:!:29.2 .±7.6 .:!:152•O2 9 25.0 19.4 209.0 334.0 372.0 24.4 1371.0 477.0 5793.0 .:!:2.21 .:!:1.67 .:!:.10.9 .:!:.21.5 .:!:19.49 .:!:.1.02 .:!:39.3 .:!:.14.57 .:!:.51.3 1U 25.9 23.3 157.u 296.0 349.0 22.5 1259.0 ~ 445.0 5508.0 .:!:2.48 .:!:2.28 .:!:12.S .:!:12.2 .:t23.7 .:t1•11 .±29.0 jfJ.77 .:t164.6 11 25.0 21.1 176.0 311.0 290.0 24.8 1205.0 454.0 4600.0 .:t2•1 .:!:.1.92 .±16.0 .:!:12.3 .:t13.5 .:t1•23 .:!:.25.7 .;l;.l2.9 .:!:.12tl.5 12 19.0 17.1 131.0 263.0 285.0 26.5 1255.0 446.0 5054.0 1.03 .94 8.06 7.71 11.69 1.41 25.6 8.5 98.7 14 31.5 32.8 140.0 197.0 372.0 23.3 * * * .±5.14 .:!:.4.53 .:!:.18.86 .:!:14.08 .:!:22.87 .:!:1.30 16 26.2 23.9 131.3 185.0 297.0 20.3 1157.U 421.0 5108.0 .:l;.3.13 .:!:2.88 .:!:.10.48 .:!:16.75 .:!:.21.18 .:t1•23 .:!:24.96 .:!:12.24 .:!:.l1S.35 18 23.8 22.9 104.0 ltl5.0 350.0 23.7 1234.0 436.0 5645.0 .:t2•1O .:!:.2.51 .±8.14 .:t14.42 .±25.25 .:!:1.23 .:!:20.7 .:!:8.25 .±223.84 20 22.6 20.9 98.0 158.0 307.0 24.4 1242.0 488.0 4875.0 .:!:.2.58 .:t2•19 .:!:7.89 .:t9.00 .:t17 •81 .:t1.93 .:!;.35. 00 jfJ.91 .:!:.164.05 22 21.6 22.4 130.8 154.0 335.0 27.0 1293.0 437.0 545Q.0 .:!;.3.0 .:!;.3.72 ;t8.6 .:!:.10.1 .:!:25. 05 .:t1•66 .:!;.39.41 .:!:.14.30 ,i68.27 * Weights not obtained 39 weight after three weeks of a's8, although t:td_s did not occur in the females. Amone the other non-lymphatic organs it was ObtHl!'ved that a loss in the relative weight of the heart ocourred although the relative i .... e1ghts of the kidneys and the livers in- Since patterns of growth are mope easily vlsuallzed when plotted in g!'"aphlc foro;,;], a detailed analysis of the changes w5,th age in lymphatic organs is presented. Body ,¥et~ht (Fle;ur:e 1). The gro\'Vtb in body weight of male and female mice is graphically prsFlentod in Pleure 1 • -• o -o -0--0- 0 _.-- M - - ... r Age in Weeks Gh.an7'6S with. Age in Body llAlr~ht in 'Ie an,; Y'Gltlolc, :.BA k1ce 40 The most rapid growth 1n bot:l sex.es occurred dU1'ing the first six weeks of life. Even in this early period of life the growth of the males was greater than that of the females, so that at six weeks of age there was a mean difference of 1.4 grams between the two sexes. After six weeks of age the amount of erowth deoreased in both sexes, but more so in the females. At twelve weeks of age the females were still gain­ing in weight, but at a greatly reduced rate, and from the slope of the curve one may assume that the females would oon­tinue to gain in body weight even after twenty-two weeks of age. In the males the gain in body weight between twelve and twenty-two weeks of age was greater than 1n the females. From the slope of the curve one may assume that growth in the males would also continue beyond twenty-two weeks of age. '11'16 sharp drop in weight which occurred at the tenth to eleventh weeks is discussed in the section on the spleen. Lymohatic and endocrine organs. The patterns of growth of the lymphatic organs, the adrenal aland, and the testes was portrayed by the use of centered two-item moving averages of the means of the absolute and relative weights. This method of treating the data suft'iced to smooth out the curves, but did not appreciably alter the trends due to age and the differences due to sex. This statist.ical method, frequently used in the study of growth, is fully explained by Croxton and Cowden (1929). 41 I06uina1 and axilla~I nodes (Figures 2 and 3). It ma7 be seen that the growth curves of the inguinal and axillar7 nodes were similar. There was a period of' rapid growth dur­ing the first six "eeks of life which tended to be slightly greater in males. Beyond six weeks, the amount of growth de­creased in females. and was followed by another period of rapid growth between the tenth and fourteenth weeks. The drop in absolute weights of these nodes which occurred between the tenth and eleventh weeks is also discussed in the section on the spleen. After the fourteenth to sixteenth weeks a con­tinued proc·;ressi va decline in the weight of these lymphs. tic organs t;l)ok place. Although SOt;'i6 slight di fferences were ob­served, the trends were, in general, the same in both sexes. A study of the relative weights indicates that the ab ... solute growth of the inguinal and axillary nodes durir:g the first six weeks of life VIas sufficient to maintain the relative Vleir;h-cs &. t a fairly constant level, deep1 te therapld gain in body we1.ghts during this 961'iod. Between six and fIfteen weeks, the absolute growth of the lJ>'nlph nodes decreased, parallellnf, the decrease in body growth. Thus, the relative weights do not show sign:i.ficant changes. Beyond fourteen to sixteen weeks a deorease in relative weights was sean. This coincided with the beginning of age involution. A sex dif­ference is suggested by the slightly higher relative weights of both inguinal and axillary nodes 1n the females OV{lr nine weeks of age. 6 IQsIJI 5 H bO .,-j r-I r-I 4 .,-j ::E ~ oH 3 .+J ..c: bO oH 2 Q) ts: 1 • .+J B:: po, 'd 0 If) III S ro 25 ~ 0 .0-- f 20 .......... • 0) So 15 ~ ~ oH 10 .+J .Q b,) .,-j Q) 5 l5: 42 Absolute J>- -_ y -0... '/ ........ ~ ~, '0...._ p-_A. ,0... , ... '0'"''''' ..... ..()' ,.. I I ,t7 I $I I ;' - - Relati ve 345 16 ill Age in Weeks Fig. 2. Normal Growth and Involution of the Inguinal Nodes. --0 M - F 20 7 V'l 6 a OJ H be or-! 5 ~ ~ or-! ~ c 4 or-! ..p ..c: 3 M or-! Q) ~ 2 1 • +> :-:-- .~ ~ 'lj p0: j 30 V'l f3 OJ H 25 b 0 0 ~ 20 "-• UJ S b.O 15 ~ c or-! 10 ..p ,.d b.O or-! Q) 5 ~ 43 Absolute ... .... - ........ s:;,-- o--..Q- .; ..; .; , , ,c , , rt' ~ - - Relative 2 3 4 5 6 7 A'!,e in Weeks Fig. 3. Normal Growth and Involution of the Axillary Nodes. - M F 44 Mesenteric nod~s (F'igure 4). The .figure shows that the changes in absolute weights of the mesenteric nodes differed greatly from those of the subcutaneous nodes. The mesenteric nodes consti tute a large mass of lyrn:,;hatic tissue ranging in weiGht from ten r.lilligra:ms a t two weeks to thirt7'-.fi ve milli .. grams at eight weeks. This mass of tissue, which grew rapidly durlnp; the first seven weeks of life, decreased in weight thereafter. The involution which was greatest between the seventh and twel.fth weeks decreased as the animals becs.me older. No sex di.ffarences were indicated. The changes in relative wei.ghts of the mesenteric nodes also differed from those described for the subcutaneous lymph nodes. During the first seven '.leeks of life there 'WEire rapid gains in the relative weights of t.').e mesenteric nodes of both males and females. Beyond the eighth week of age there fol ... lowed progressive losses in. the relative weights'; these losses coincided with the actual age involution of these nodes In' both sexes. The decreases in relative weights were greater than those in absolute weights because the body weights con­tinued to increase. Similarly, the fact that the relattv6 weights were higher in females than in males was most likely due to the grenter body weights of the males at comparable ap;es. ?bymi (FiF~ure 5). A rapid prenatal and neonatal absolute growth 'Was indicE', ted by tht<} large absolute weights of the thymi at three weeks of age. Between three and five weel{s • ..j..:l 200 ~ • Cf.l c§ o o rl "- 80 60 40 20 80 45 1 2 3 4 5 6 7 8 9 10 12 Age in 1;Veeks Relative , 14 16 " '0----0 18 20 Fig. 4. Nornal Growth and Involution of the Mesenteric Nodes. 46 there was only a slight continuation of growth of the male thymi and age involution became eVident between five and e:tght weeks. In contrast, the growth of this organ 1n :temales continued up to six v/eeks of age before involution w.1th age began. The involution was at first slow, but be­came more rapid, especially between twelve and eighteen weeks of age. There appears to have been a slight decrease 1n the amotLYJ.t of involution in the oldest animals. The greater ab­solute weights of the female thymi may be aCcotUlted for by the lonzer period of growth (up to six weeks) and probably also by a faster rate of growth in the very early stages. The difFerences in absolute weights between males and females were present throughout the period studied. :£<ligure 5 shows that there was a loss in relative weight of the thymi even in the very youngest animals. The amount of loss, however, followed closely the increase of body weight. 'l'hus, it was ra.pid during the first six we(3ks, gradually de­creased between the sixth and the fourteenth weeks, and vir­tually ceased in the oldest animals. Correlated with the greater body weight of the male thymi and greater absolute weights of the female thyrn! \Vere the greater relatIve wei~~h'ts of the female thymi throughout all the ages studied .. Spleens (Figure 6-1. The growth curves of the spleens were very similar to tho~e of the suboutaneous lymph nodes. There were rapid gains in weight for the first six weeks. These were followed by decreases in growth between the sixth 70 ffl 60 E as H ..b-DI 50 .-l ,--! ..-I .'.5.....!.. 40 !:l or! +J 30 ~ b.Q or-I <D F: 20 10 0 • +J :=: p., 'd p0:) 600 ffl S HOJ 500 eJ 0 0.- l 400 .......... • ffl ..EC. D 300 ~ """ !:l o,-l 200 +J ..d tlD o,-l <D 100 •?. ~ 47 Absolute --- M - - - F , , ~, '0---0... ..... --.. ..... ~ ... 'll... ....... .. Relative ~­- - -.0-. - - .-0- 1 2 3 4 5 6 7 8 9 10 12 14 16 18 Age in Weeks Fig. 5. Normal Growth and Involution of the Thymus. ----0 20; 48 and tenth weeks. After the tenth week there were further in­creases in growth which continued to the sixteenth week. Be­tween the sixteenth and twentieth weeks there was very little further growth. The depressions in weights which occurred in both the spleens and subcutEtneous nodes during the tenth and eleventh weel,:s is inexplicable. It is suspected that the ani­mals may have been subject to some type of "non-sDecifie stressfl by some unknown environmental factor. This apparently affected the males more than it did the females" since at this age the mean body weights of the males dropped even below those of the females and it may also be noted that the spleens of the males were heavier than those of the females except c1urin.!:'\ the tenth week. The changes in the relative weip:hts of the spleens were similar to those 1n the subcutaneous nodes. The re 1a ti ve weights fluctuated widely but def:i.ni te downward trends with age were observed in both males and females. As 1n the lymph nodes, the absolute growth of the spleens during the fiI'st six weeks of a.ge was great enough to keep pace with body growth and therefore the relative weights were high. However, as the anirlals aged the absolute growth of the spleens tended to lag slightly behind that of the body and this resulted in the trends toward a slight loss 1n relative weights. Adrenal glands (Fit:;jurc 7~. Definite sex differences in the absolute and relative weights of the adrenal glands were observed. 'Ibers were periods of rapid absolute growth which 80 70 60 50 40 30 20 10 • I I ,6 ,, I l' I I A, JJ' " '<:1"'''' 49 p.- ,. "" Absolute __ -0 ,tJ- - -..0- - -..0- M - - - F .p ~ O~-L~~~~ __ ~-L __ ~~~ __ L--L~ __ ~-L __ L-~~ __ ~-L~L-~ __ I7l S til ~ o o ,--i "-.. • I7l 6b 50 t.t;~ F< • ..-1 200 Relative Fig. 6. Normal Growth and Involution of the Spleen. t1.l S 0.1 H u: oM 5 r-f r-f oM ~ ~ 4 • .-1 +> ..c 3 bO oM Q) ~ 2 1 ~ o ,:q 30 rn E <Xl 2J o o r-I 20 "-... • rn .,~... "'" 10 50 , ct Absolute. ;0- - - -0- __ 0-" ~d' -- -0--- . .......... ...... 0 M - - - F Relative 1 2 3 4 5 6 7 8 9 10 12 14 16 20 Age in Weeks Fig. 7. Normal Growth and Involution of the Adrenal Glands. 51 lasted six wee!{s tn the males and seven weeks in the fen181es. Ho further weigb.t gains ocourred in the adrenals of males, but in the fernalos they continued to ga:n weight up to tV/e1ve weeks of age, bey·nd whioh their growth virtutllly oeased. Associated with the above differenees in ",bsJ:tute weights were the differ­enoes in relative weights. In the females the re1a.tive weights remained fairly oonstant, indicatlng,that absolute growth of the adrenals kept 1:)ace with boC;:y growth. In contrast, a definite trend toward a loss In relative weight in the ~ales was obs62ved even 1::1 the earliest stages of life. This tendency to decrease did not extend beyond fourteen weeks of' age. tfestes (Fio.;ure G~. The growth curve of the testes shows that the absolute growth of the testes followed the pattern of overall body grC"4~h.. The most rapid growth occurred in the first eight wec:~f3 or life, beyond which the growth decreased and there followed a gradual leveling off of the growth curve bey-cnd thF.) fourteenth week. T'n.e decrease in testes weight whIch occurred at ten weeks of age is probably associated with the similar decreases which occurred in the spleens, subcutaneous nodes, and body weights at the same age. The curve of relative growth shows that there was a rapid increase in relative weights up to flve Weeks, followed by a period between five and tan weeks during which the absolute growth was just suffioient to keep the relative weights constant. After ten weeks the relative weights tended to decrease. tI.l E ttl ~ bO ori rl rl 'r-! ~ C • ..-1 +l ,.C be 'r-! CD ~ • +l ~ .& 0 CQ tI.l E ttl H 0 0 0 rl '-..... • tI.l §; ~ c ori .p ,£j M .r-! CD > ~.~ 60 40 20 100 80 60 40 20 600 500 400 1 52 8 9 10 Age in Absolute Relative Fig. 8. Normal Growth and Involution of the Testes. M 53 3. Summary and disoussion This :)hase of the investigation is a study of the changes with age and the sex differences in the body weIghts and in the absolute and relative wein;hts of varlous lym;)hatic organs and the adrena12l, testes (in the male), kidneys. hearts, and livers in CDA mice. It was observed that the first six weeks of life marked tIte peri-ld of most ra~).1d growth of body weight and that beyond this age ther-8 was a progressive reduction in the Growth of both males and females. 'I1J1e reduction in growth was more marked in the females and this resulted in the marked sex dif­ferences in body weights of the adult animals. It was also noted that even at twenty-two weeks of age males and females were still increasing in body weight. All the organs studied grew rapidly during the first six to eight weeks of lIfe, but after that the growth of lymphatic organs differed from the growth of non-lymphatic organs. A defini te c)I'Oi1resslve loss in absolute weights began to take -place in all the lymphatIc organs except in the spleens at some time during the period covered by this study. This so-called "aBe or phYSiological involution" appeared earliest in the thymi, next in the mesenteric l;ynrph nodas, and last in the subcutan-eous lymph nodes. The initial rates and periods of growth also differed among the lymphatic organs. The thymi apparently grew faster but for shorter periods of time tha.n the other lympha.tic 54 organs; thus before the third week of neonatal life the thymi of males and females reached their maximum size and age invol­ution began. The mesenteric nodes grew rapidly for eight weeks and then suddenly began to lose weight. T'lle subcutaneous l)'"W~)h nodes and s}:;leens grew rlrpidly for six weeks, then less rapidly for a.nother ten weeks. At sixteen weeks of age the .subcutaneous nodes but not the spleen began to undergo involu ... tion. After this age, however, the spleons did not continue to increase in size. A sharp drop in the absolute weights of the subcutaneous l'Ylnph nodes and s;>leens was observed between the eighth and thirteenth we(':ks. Similar drops were observed in body weigh~s and absolute weights of the testes, and the suggestion WtHJ made that these drops mi;.?,ht have been due to a "non-specific stress". The only real sex differences in absolute growth were found in the thymi, whLch were heavier in the females, and in the spleens, which were heavier in the riales. The·adrena.l glands of the females increased steadily in absolute weights up to twelve weeks of age. Those of the nalns reachod a maximum mean absolute weight at six weeks. After the maximwn we1ghts were attained, there was little variation in the weights ()f these glands during the rest of the ?eriod studied~ The testes showed rapid absolute growth up to six weeks of age, followed by a gradual decrease in growth up to sixteen weeks. This was the typical growth pattern observed for the 55 hearts, livers, and kidneys. The data thus far presented suggest a correlation between the n;rowth of the body as a whole and the growth of the l~m~hatic organs, adrenal glands and tastes. The "/:)rk of Chiodi (lS!38) J Plagge (1941) and Rape 1a (1944) shows that the effect of' sex hormones is to cause a decrease in size and weights of the thymus. Similar effects have been reported for the secretions of the adrenal cortex by Rape1a (1944). The most rapid growth of all lymphatic organs as well as non-lymphatic organs occurred during the early period ot1ife. During this period the testes and adrenal glands wera also still growing, and it might be postulated that tbeir s€'lcretor:,r activities were low and thus there was little tissue restraint on the p,rowth of l;;rmphatici, so that the lymphatic organs gained weight ra~.1idly. There is much physiological and anatomical evidence to support the assu.>nption that the testes function is low Qurlng the early period of life. However. there is no way of deter­mining, either directly or indirectly, t1.1e levels or kinds of adrenal cortical hormones in an or!~anism at any age. 'rhus, the aSSlllTIlJtion the t the ad1."enal glands are not actively secreting during the period in which they are growing is without support. It nay further be noted that between six and eight weel{s of a;Ie the adrena1s and testes rea.ched a size 19.r"',e enough to per:mJ t the ass:J."'1ptlon that they were act! vely secrettng t 1"jeir respect! ve hormones. During this 56 period involution 01' the thymi and mesenteric nodes began and there was a reduction in the r~rowth of the spleen and subcutan,;olJ.s noue Ct. Involution of the subcutaneous nodes (lld not:e!jin until the s:l.xtsGr.th lJH,ek, when the teshlS had attained their full size. It thus saeelS that among the factor's controlling th<l growth of lymphatic tissues, the dynandc interplay of growth hOI'1-:10ne, aiipe·;.&.l cortical hormones) and sex horrn()nes r.lsy be a d01~linant one, and the. t there is H J.ifference between the sensitivity of the tihyral, 1:16SentsI':tc IJrmph nodes, BubeutHl1EJoUS lymph nodes, and spleens co thE! [lOr-mones involved. 'rha t 'the hC:t'f.lOrlEH' in the fe."lal€!s do not arfsc t 1 *hJ'Inha tic ,I "0. tissue as much as do the hormoues in the males :ts suggested by the largE~!' absolute weights of. the femEtle thymi and by the fact that the relative weights of all the lymphatiC organB tonded to be higher in fElInales. 57 B. Changes in Body Weights and in Weights of Lymphatic and Non-lymphatic Organs of CBA Mice Four Days After Adrenalectomy 1. Introduction The results reported by other investigators indicate that the secretions of the adrenal gland cause an atrophy of the thymus whereas adrenalectomy results in hypertrophy and hyper­plasia of the thyr:ms and other lY;::1phatic tissues. However, systematic studies of the effect of adrenalectomy on the growth of the thymus, spleen, and lymph nodes have not been reported on any animal. It therefore seemed important to conduct such an investigation by observing the changes in the absolute and rela ti ve weights 0 f lymphs. tic organs following adre::lalecton:y. It was also hoped to further elucidate the influences of age and sex on the lymphatic tissue-adrenal 1'19 la tionship, b:;r perfOrlJing these experiments on animals of prepubertal and early pubertal ages. 2. Methods Five male and five virgin female CBA mice were adrenal-ectomized each week between the third and twelfth weeks and sacrificed four days later. These mice oame from the same colony and had been kept unde.!:'" the same concH tiona, as those of the normal group desoribed in the previous experiment. The anirnals were ac.renalectomlz(;)d four days before the day on which the normal groups were to be sacrificed and thuB the normal and adrenalectomized animals ~vere of the same age. 58 3. Results Body weight. Adrenalectomy resultea in losses in body weight in both males and females. The amotmts of actual and relative loss are presented in Table 5 and are graphically represented in Fir';'lll"e 9. .i\f5. Normal 2 wk. 6.1 3 7.6 4 13.1 ,5 14.6 17.0 7 18.6 8 21.6 9 21.0 10 21.0 11 21.0 12 23.3 UA1ES Table 5 Losses in Body Weights of Male lie Female aDA Mice Pour Days After Adrenalectomy .. . fmAL.BS Adran&!- Aotual % Ad.renal-eotomizeCl Los. Loss Normal eotomized 6.1 5.' 1.7 22.4 7.8 7., 8.7 4.4 33.6 11.3 8.3 12.6 2.0 13.7 1).1 11.8 1).5 3.' 20.6 15.6 11.3 12.9 ~h7 30.6 16.1 11.9 16.9 4.7 21.8 18.6 14.3 14.6 6.4 30.5 17.3 14.2 19.1 1.9 9.; 18.0 15.5 14.9 6.1 29.0 17.6 15.2 18.4 4.9 21.0 19.1 1).7 23,2.7 Actual Loss .3 3.0 1.9 4.3 4.2 4.3 ).1 2.5 2.4 5.4 Average 2).7 Average % Lo8s 3.7 26.5 13.9 27.6 26.1 2).1 17.9 16. it 1. 15 .. 8 g8.l 199.0 19.9 Examinatio,n of these data shoVis that the actual loss in body weight was significantly greater 1n the oL~er animals and that it tended to be greater in the males than in the felnales. On the other hand, the loss per one hundred grams body weight was as great in younp; animals as in old animals. The amount 59 6 It; r- " !':' : " 4 - .~: ., " ~ " " " w '~ . , , , ~' " . a 4 : :, ~: ttl - ~: ~ .. :~ , " , r:r :~ . ;: " .... -- " '",' , '.~ 1.) I " ' .. " ,.Cl " . ... b!l 2 :"l , .,..j - ,,: ... " ," ~ " ~ r:" :. " !!! , . " -: " " G-l '. . 0 10- :j, , : i'Il , v.l . . . 0 0 ::h " , H :5 4 5 e 7 8 9 10 11 12 Age in V{eeks Shaded Bars M Clear Bars F 35 - ~ • 30 - '::': 1.) ~1 1"11 ..". ,I - r'u.. ': " M 25 :~- d - ':i ~ : :1 " '. ~ r-: \1 : z0 20 ~ " : I"; c..; r- " ~ .. . 0 " : " " "<R 15 " .. I- " I'll " " a;s ~ : ::'11 ", " " : " • 10 " ' , " :+f.:l f-- " l>: : r-: " \' ~ , " " t: " "<"'I G ~: " " ~ , , .. : CI'.I " " ',' t' : " '. 112 '. '. ., Q :,j~ 1-1 0 " " , , 3 1: 5 6 7 8 9 10 11 12 Age in 1NOGks Fig. 9. Loss in Body Weight of Male and Female eBA Mlce Four Days After Adrenalectomy. 60 of" loss fluctuated widely 1n both sexes but tended to be slightly higher 1n the males. Ly:t'llphatio ors.ns and k1dnez. 'fhe absolute weights of the various lyrnphatic organs and the kidneys f"cur days after adre:1alectm;lY in male and female mice aged three through twelve weeks are compa.red with the normal weights of mice of similar ages (Tables 6 and 7). After adrenlaectorny there was a redUC­tion in the absolute weight of the kidney in animals of both sexes, but there were significant increases in the absolute weights of all the lymphatic organa, except the thymus, espec .. ially in the older animals. It has been shown that the normal absolute and relative weights of the various lymphatic tissues vary with a:;;e. It was therefore decided to stUdy the amounts of alteration in at.lsolute and r'elHtlvo weights fol1ow:L~g adrenalectoMY, by calcula tinp; the ')erc~)nta:e of d:i fference from t.he normals. The percentage of change following adrenclectomy was calculated for each organ at each af:;e a.nd frora these were determined three-l tom movin,,; averages which are represented in , the i:.;raohs (Fizuras 10 and 11). Thls method allowed a better anal;:,sis of the trends in the amounts of increase in absolute and relative wei6;hts followIng aurenalectorrry, as influenced by sex and a";6. Inguinal and axillary nodes (F'igures 10 and 11). The growth of the inguinal aud axillary lymnh notles follow5ng adrenalectomy was vor'Y sitlllnr and these (":lands may he • 'IIA g.e - in Wks. 3 4 5 , 7 i 8 k tI 9 10 11 12 3 4 5 td • 6 i'"f ..0.. 7 () ~ 8 i t: 9 ~ 10 11 12 61 Table 6 Average Body & Organ Weights in Male CBA Mioe, Intact and at F01.tt» Days After AdrenalectGmy BodJr ;ng . Ol'{5a.nWeiiht."in iLiie • Wt. Ax. Mes. in Gms. Bod. Nodes Nodes us S 1 en 7. 2. 2.3 .. 45. 2. .3 .;t.43 -+.449 .;t.3l .:t1•14 .t2.39 .:t2.41 1).1 3.1 2.8 19.2 49.1 47.' .;t.44 -+.)1 :t.21 -+.61 .±.l.SO :t2.97 14.6 ).6 3.4 21.2 50.4 55.9 -+.36 .t.41 -+.43 .1.83 -+2.88 .;t7.15 17.0 ;.1 4.) )1.2 51.6 79.2 .t'-.25 +.39 .:t.:31 .t1•6S .t.4.47 +6 .. 67 5.0 - 18.6 4.0 34.7 42.1 50.7 +.77 +.34 .:t.42 +2.63 .t.3.22 +2.6) 21.6 '5.6 4.9 3,.8 53.7 lrO.2 +.03 i·53 .:t.47 +2.08 .12.30 +6.65 21.0 - - .5 3.9 32.9 42.7 72.1 +.62 .:t.31 .:t.06 +2.08 .:t2.)6 +4.09 16.5 5.4 4.2 -25.3 40.2 !6.S +.64 +.)4 +.27 .11.47 +1.96 _ .78 21.0 - - 3.7 ).1 :u.8 39.2 51.5 2,:3t..7)5 -4+..4)1 i+..a2 ; )i01.7.; 7 .4:t14..);9 .g1e.l,1 .1.59 :t..47 i·29 it,2.29 .:t2•16 .t. 1.64 i.3 4 2&.0 ,., 2.0 4.9 51.0 +.20 +.16 +.20 +.47 .±.l.)8 .:t4.57 !'.7 '3.4 -3.2 14.6 53.1 40.2 .t..41 -+.27 .:t.25 :t1.53 .:t2.9) .;t8.19 12.6 3.) 2.9 29.1 54.2 53.7 1.3.:t..591 :4t...4)1 -+ 14,1.525 .2:t 54.8.3 ) .7t76..547 5+82..324 .:t.37 -+.49 +.21 .:t1.77 ±2.79 +7.07 12.9 4.3 1.7 23.5 51.1 58.1 +.46 +.7l +.86 j:).68 +1.48 ~.41 1~.96 7.3 '5.7 34.5 '48.e .9 .1-55 .:tl.l +.63 ,tl.4° .:t,5.82 .:t.4.38 14.6 5.4 4.6 44.4 53.8 55.1 1+9..414 '+5..66 9 5+..66 +6.15 .:t2.79 tl·68 -35.2 49.8 4.3 .:t.92 +1.02 +1.18 .1').79 .±.l.43 +4.23 14.9 -6.6 -5.9 26.98 :n.6 ~8.2 +.72 +.75 +.77 +2.23 +2.36 .:t12.46 1'S".4 l.2 t.l 36.8 38.0 94.1 .:t.54 -+.58 .:t.78 ,:16.87 -+6.12 -+6.04 105. -+6.88 193.0 .t9.17 222.0 .t12.99 261.0 .:t14.9 311.0 +1).6) 1'61.0 .t.12.35 375.0 .ft l.43 248.0 .t.14.54 )03.0 '1'14.5 347.0 $0.5 87.0 .th03 121.0 .±5.12 194.6 +14.96 206.6 ~.06 1 7.1 ±11.79 267.0 +22.61 206.8 -+10.1 274.3 !,lO.76 232.0 .t15.12 276.0 -+28.01 62 Table 7 Average Body &: Organ Weights 1n Female CBA Mice, Intact and at Four Days After Adrenalector:!y Age Body OrS!n WeiQht. in !seas in ~gt. Ina-. Ax. Mes • WIts. in Oms. Itod •• • d.e. Nodes tlKema Siae.,n ICid.ne, g 3 7.8 2.~ 2.5 9.3 47.4 30.1 101.0 -+.29 -+.63 .:t.24 .1.84 s.,2.43 ;t2.11 .;t4.35 4 11.3 1.9 2.1 22.6 54.2 31.2 16).0 -+.17 -+.21 -+.17 .12•61 ,;t3.1 -+1.97 j;.l.88 5 13.1 4.6 4.0 23.6 61.8 53.3 186.0 -+.48 -+.33 -+.39 .11•21 -+2.72 .:t.4.39 .:t.6.45 6 15.6 3.7 ).0 28.8 68.7 59.9 223.0 .t. .64 .;t.30 -+.30 .t.2•O .t.2.97 s.,4.68 .t.14.96 ~ 1 16.1 5.2 4.2 31.4 54.7 48.9 225.0 -+.58 .;t.5" -+.60 +2.39 j:3.86 .±4.02 .;t10.2 8 18.6 ).5 3.5 34.3 57.0 52.1 247.0 .;t.08 .;t-29 -+.36 .:t2.55 .t.1-3' i 1•67 .15.1 9 17.8 4.7 3.9 33.7 57.5 68.0 242.0 .1.39 i·24 .1.34 .t1.98 -+2.69 j:3.09 -+6.82 10 18.0 4.7 4.3 28.5 53.5 63.4 221.0 i·36 .t.48 .t.45 .;t2.62 i 2.59 i5.01 i5.25 11 17.6 4.3 3.1 31.1 54.4 51.1 212.0 i·39 i.32 -+.28 ,tl.14 i 1.94 ;t..l.06 .t'.93 12 1.9.. 1 4.0 .1.3 25.0 50.1 54.1 231.0 ~~ 12~ .20 11~6 1,21 2·I' 6.1~ 3 7.5 ).1 3.5 8.5 65.5 52.6 107.0 .t.56 i.33 .t.21 .11.79 .14.91 .±9.39 i 6.45 4 8.3 2.9 3.) 13.8 51.9 35.9 112.7 .1.21 -+.43 i·29 .±..51 £3.34 -+4.28 .±.5.61 5 11.8 5.0 4.4 27.1 48.9 66.8 175.8 • -+.52 -+1.0 .±..85 J:,l.13 .14.33 -...a. US ~.26 I 6 11.3 4.8 4.4 22.1 59.0 58.7 152.0 i +.27 +.32 i·37 i 1.99 .11.19 .;t6.94 jfJ.53 .3 - - '0 7 11.98 5.8 5.9 23.' 64.2 61.3 159.6 CD +.16 +.62 +.77 +1.89 +4.19 .:t15.54 j;fI.51 1 - - - - - 8 14.3 4.8 4.7 )9.3 57.9 66.1 119.0 .t.26 i·34 +.37 .t4.11 +2.36 z.2.45 +1.54 ~ - - - 9 14.2 7.23 6.1 38.4 60.1 80.9 -+.53 .±.1.0 -+. • 88 .±l.10 i5.73 .17.49 10 15.5 6.7 5.9 )2.1 54.6 53.2 197.9 .;t-51 .;tl.08 .t.90 .t2.32 .14.57 .±.4.18 .14.02 11 15.2 6.9 6.4 48.8 41.9 78.0 187.3 .:t.48 -+.80 -+.39 .:t2.41 -+6.88 .t,9.09 .17.85 12 13.7 4.98 '.9 38.2 48.4 70.6 180.6 i·19 -+.95 z·76 .ta.35 i5.41 -+8.91 i 2 •04 63 H 160 __ -----------------------------------------------, CI.1 f o :;;;; 150 I-- f1 ..... ~ 140 t-- "(j CD ~ 130_ E o o ~ 110_ ~ :: , I"" TI :. :: . d 100~----~~--~~~~--~~·~~~--~~~·----~ : lli R tD a 90_ ~ o ~ GO~ ______________________________ --------------~ 4 5 (3 '7 8 9 Age in Weeks 10 11 12 Shaded Bars M Clear Bare F :Joo ~----------------------------------------~ • ~ 180 r- "cCr•D. 160 r- .:..:.'' ~~ 4 1:"". ' . 5 8' '7 8 9 Age in Weeks ... .. 10 11 12 Pig. 10. Alterations in Absolute and Relative Weights of Inguinal Nodes of Male &; Female CBA 1\11oe Four Days After Adrenalectomy. I""f I ~ 0 ~ 160 - .~.... ~ 150 I-- "t:I G) ~j), 140 I-- l.1 0 ,... 0 , 130 t-- +l ~ ., 120 0'1 I-- ~ ,::; 110 ..... ~ 4) tlO ~ 100 ~ . ~ ~ 90 I 4 200 r- • +l 180 ~ ~ .. I""f 4) 160 I-- tx: ..~... 140 Q) r-- fIl d III': Q) 120 . ~ t-- . () ,::; I-t ~ 100 4 Fig. 11. 64 ... ... Ii ~. r:'! "" " ~ :: " r. .."" ~. I I I I I 5 e 7 S 9 Age in Weeks ... ... ~ 1:'1 r:: . , r=: , : r:, ' .. ". ~ ' . . 5 6 7 a 9 Age in Weeks r: · • · I 10 Shaded Clear F. · 10 r,: ... ~:, .. , .. . " " , [.;. . I n Bars Bars r.:: " . 11 M F Alterations in.Abso1ute and Relative We1shts of Axillary Nodes of Male & Female CBA Mice Four Days After Adrenalectomy 65 considered together. A definite sex difference was obse~ved. In the females theps was an increase in the absolute weights at all ages, but the a.moullts of increase tended to be greater in the clder animals. In the younge t ;males there was either no 0:ain or a loss, but after seven weeks of age the amount of ga.in in the ingu1nal and axillary nodes increased. At eleven weeks 01' a:;e the incrs!:Jsnt found in nlf~les more nearly H99roximated that observed for females. Shiller rGsults were observed for the rates of increase in relative weights. There Vlere significant increases at all ages in both sexes but the amounts of increase wer& greatest in the oldest animals. The final relative weights were greater in females than in males at all ages but the greatest sex dif­ference was observed in the younger age groups. It is interest ... ing to note that in young males losses in relative weights failed to occur daspi te tb,e losses or insignificant gl, ins in absolute weight. This was due to t116 large losses in body weight which occurred ~fter adrenalectomy. !{esenteric::odes .(Fi?jUre 12). The reaction of the mesen­teric: nodes to adrenalectomy was somewha.t dIfferent than that of the subcutaneous 'nodes. There were losses in the absolute we1.ghts of the mesenter:ic nodes in the young of both sexes, but increases in ttte animals over el;:ht weeks 0 f ar~e. The transition frOl;\ the period of negative to positive changes occurred abruptly between the ap;es. of seven and nine weeks. A real sex dlfference VIas not 8.pparent, but the data suggest 66 t""i IX! 150 ~ ~ .0 140 f-- .p .,..j :t: '0 130 i-- \l) r- ~ til , sp. . 120 - 0 0 • 110 +! - '=' " ~ " " • l/) .0 100 -< a n " " 1:: [ -- .. ..... 90 '- ~ ':. i- ID til s::: ~ 0 " -'f. • .p Z~ 80 70 200 180 :! 140 IX! \l) ~ o s::: 120 H 100 - - - - - - I I I 4 5 I';" " l " , ::h I I I I 6 7 8 9 Age in Weeks I I I 10 11 12 Shaded Bars M Clear Bars F ::r- ~ ,". " ~ m r- " " ~ ". :;: 3 4 5 6 7 8 9 10 11 12 Age in 'Weeks Fig. 12. Alterations in Absolute and Relative Weizhts of' Mesenteric Hodes of Male &: Ferna'le CBA Kice Four Days After Adrenalectomy. 67 a larger increase in the fel:1ales of the oldest aga. The changes in relet! va weights 0 f mesenteric nodes after adrenalector:1Y were also i:;dicatlve of a genera.l trend from smaller gains In young animals toward greater gains in the older animals, with the ,o:reatest amount of gain in the oldest females. Thrnus (Figure 13 ~ • The absolute growth of the thymus following adrenalectomy was entirely dlfferent than that at tILe other lymphatic organs studied. There was an increase in the absolute weight at all ages in both sexes, but, with the exception of the youneest anic'1s1s, the increase was greater in the r:18les. In the young males the amount of increase became progressivel-y greater up to six weeks of age and beyond that age the amount of increase became s~laller as the animal became older. In the fenales the amount of increase did not follow e.ny defin:l te trend during the first six weeks, but beyond this age there occurred the same proc;r'essive decrease in the amounts of gain as was observed in the male. The increases in releti ve weights were also greater In the T;lsles than in the fe:i'J.alea and the same tendency for the increase to be greater in the younrv,est animals was observed. Spleen. (FiHure 14). A pronounced sex difference was ob­served in the chnnees in the absolute welghts of the spleen followlng adrenalectomy. In the females there was an increase at all ages, but in the males there was a decrease which was rC: .;.J 130- <rl $l1: 'lj CD ~ ttl P. ~: o 120 I-­u • I1J ~ 110 I­~ oM 4J r 68 . !-: :'1- r'.:.'' !':.. :1- til ••. ,. 01 ~ 100~------~~--~~~~--~~~~ __ ~~n~ __ ~~~~~ ____ ___ 3 4 5 6 7 8 9 10 11 Age in Weeks Shaded Bars M Clear Bars F 200~--------------------------------------------~ • 4.l ?!: 1S0- ....•. Q) m 160- .,r..:. CD 140- Cf.l Ctl \-,.r ~ g 120-- H 4 5 .: I- 6 7 8 9 10 11 12 Age .1.n Weeks Fig. 13. Alterations 1n Absolute and Relative Weights of Thymus ofM:ale and Female aBA Mice Four Days After Adrenalectomy. r-I 1E.:1 150 ~ 0 z :S 140 ~ or-! ~ 'C 130 r-- \Ii ~ ~ p.. f: 120 r-- 0 0 • 110 -~.. I-- ::lCO- • m 100 ,0 '< j:I .,..; 90 .: r-- ~ II) til j:I 6 60 t-- '\.. ~> 70 I I 3 4 n .~ :: :. ~ I 5 69 . :'r- ~ .- I .- li t ... J 1 J J 1 1 e 7 8 9 10 11 Age in Weeks Shaded Bars M Clear Bars F 1 12 200 __ ------------------------------------------------~ • .p ?r: 180 r- • r-I ~ 160 r-­,::: or-! Q) 140 r-­\' I} OJ ID t 120 ~ ,::: :. " H ~.:. ~ ~.' ~~ 100~ ______~ ~~-~~~~.. ~ ~~~~~~~~~~~--~~----- 3 4 5 6 7 8 9 10 11 12 Age in Weeks Fig.. 14. Alterations in Absolute and Relative Weights of Spleens of Male and Female CSA Mice Four Days After Adrenalectomy. 70 greatest in the youngest animals; this decrease became less and the older animals showed so e increase. No real age difference was observed in the females. Examination of the changes in relat1ve weights shows that in the maies there \vere increaSes in the amounts of gain as they became older, while in the females the amounts of increase were almost equal among thevaricuB ages. 4. S'.mnnary and Discussion k It has been shown that the adrenal gland plays an impor" tant role in the regulation of body growth as well as the growth of lym::>hatic tissue and also that its influence on lympha tic tissue is modified by factors of age and $eX. The overall metabolic changes which occurred durIng a four day period f'ollow5.ng adrenalectomy l6d to losses in body weight which in terms of absolute loss were greatest in the old anImals but showed no real age differenoe when rela ted to normal body V1eieht. The amounts of loss were slIghtly sreater in the r.:ta1ea than in the females. It thus seems that. within the age grou!'s studied, the adrenals exert proportionally the same influence on the overall body growth" or on the factors controlling body growth, in young and old animals, but that thJs influence is sl1..ghtly more pronounced in the mal es. In contrA st, adrenalectomy l-s,d to a1 tara tions in absolute and relative wei;:;hts of the 1ymp'cl.atic organs whicb differed in type and amounts according to the sex of the an:i. 'Hils, the 71 age at the time of ablation, and the particular lymphatic organ. Thus, the ingu.inal and axillary nodes of the young females increased in absolute and relative weishts far more than did those of younC males.. In the la tt61" ttleIte were even losses .. Increases in the absolute weights of the spleens of females occurred at all.ages, but decr>easos occurred iu all but the oldest males.. The mesenteric nodes in Iasles and. fema.les of less than eight wee:·;s of aGe decreased in absolute weight, whereas in those ovor eight wec 1:s there was an increase. The increase in absolute we:!ghts of Lbo thymi after adrenalectomy was entirel-;,7 dU'f'erent from that of the other lYlophat1c organs .. In 'i1~~18s and females over six we/aks of llr:S the trend was toward progre8~ive decreasGS in the amounts or gain RS the animals became oldor. The differences in th,e growth of subcutaneous lymph nodes, mssFJnterlc nOdes, thymi and srleens [',fter' adrenalectomy sug-gest a possible correlation with the differences in tte normal growth of the adrenal glands. In the study of nermal ,~ov:th 1 t was found that beyond six weeks of age the adrenal glands of the females wero hes.vim:o t.han thoso of the maLH:I a11.c1 t.hat the sub­cutaneous lymph nodes of the femalos tended to be slightly lil1'hter than those of malos.. However, after adrenalectomy the SUbcutan­eous lyY:iy)h nOUDS 9f the ~~e11lal's ::,q-:ned ::lore than t:l.Gse of the males. In :'iorc!81 anlT:1s1s the s,,:lcfms, 111m the Ij'luph ncc~es, tended to be sl~ ghtl;/ 11.c;hter 1n the female s. After 72 adrenalectorry the spleen of the female showed the r-:reatest increase in weight. The norr.:lal mesenteric nodes did not show any real sex differenc~s excent in the oldest animals. A sex difference following adrenalectomy was only sug;~'ested and thJs only in the oldest an:h:1als. It may further be noted that in these nodes adrenalectomy during the period of tbelr ;':'lost rapid growth (prior to the eighth week of aise) resulted in an actual loss in absolute weicht, Where!lS E'lfter eight weeks of ap;e adrenal­ectomy resulted in an increase :1.n absolute weight, thus indi­cating a role played by the adrenal in alSe involution. In the case of the l~rmph nodes and spleen, as the anh::als beca.me older ~nd the adrenflls becar:1e larger there was a decrease in the growth of these glands. However, after Qdrenalecto~y these glands oxhlbl ted co ntlnued growth, particularly in the oldest animals. To a carta the mesenteric nodes. extent this was also true in In reIn tion to the other l)'1tlphnt Ie organs, the th.ymus was distinctly at;-pical i.n its nor.(!tal ;-:;rowth and a.lso in its reaction to a.drenalectomy. The normal male thymi were lighter than those of the fer.lales ti'. t all ages. Follow:tng adrenalectomy the male thYl:1uS showed the grea.tf:lst percentage of increase. This Vias in exact opposttion to what was observed 1n the lyml)h nodes and s:)leen, wb.er the female showed the greatest increase. F'tlrther, in the thymus the tendency was for the gros. tea t per-centage of:_ncreasc to occur in the you"ng anirlals. 73 The study of the changes 1n relatIve weights following adrenalectomy showed results whicIl, in general, agreed with the results summarized above • . It may thus be concluded that: (1) The growth of lym­phatic tissue Is directly rela.ted to the function of the a.dre­nal glands, but is further influenced by factors charaoteristic of age and sex. (2) The age dlfference in the response of lymph nodes, spleens and thymus to adrenalectomy seems to be partly related to dtfferences in the activities of the gonads and adre­nal r;lands in prepubertal aniY')als as contrasted to those of pubertal sni:!mls. (3) The sex difference 1n the res-ponse of lymphatic organs to adrenalectomy at anyone age seems to be relatod to the sec'~etory aetivl ties of the gonads and perha.ps also to a sex dlffel'ence in the adrenal glands as evidenced by a sex dlfference in their sizes. (4) There is a difference in sensitlvity among the different lJ111phatic orga.ns to the lack of the hormones of the adrenal glands and probably also to the other hormones or combinations of hormones. Thus, any studies concerninf~ the facto:rs which affect the morphology or function of the l~nnphatic tissue r:'!ust include sar:r~::>les of the thyr;ms, spleen, and lymph nodes of both sexes. 1. Introduction 74 c. The Normal Growth ot Lymph Nodes, Thymus; Spleen, Genital Tissues and Adrenal Glands 11'1 Mice from Six to Sixty-two Weeks of Age The re~mlta of' Experiment B showed that there was a oor-relation between the overall body growth, the growth of the adrenEtls and teste-a, Rnd the growth and involution of lymphatic organs. It was also shown that the increase in lymphatic tissue weight, fullowing aurenalector.1Y, was greater in femAles than in rnales. Further; it was observed that the amounts of inorease in weight a.fter adrenaleo tomy were influenced by the age at which the operation was performed. Since the results of these investll;:atlons apply only to animals of prepubertal, pubertal, and early .mature ages, it was felt that it would be profitable to conduct similar studies on animals of later mature and senile ages. Accordtng1y, the first phase of such a project was the study of the normal body growth and growth of lj~phatic and eenital organs from prepuberty to senility_ 2. -Procedure '111e an' ~':lals used In th.is study were from the same stock as those used in the two previous st.udies. However, due to limitations of space and the supply of breeding boxes, certain modifications in their ca.re had to be improvised. Ten males and thirty females from the old colony were selected as breeders. These were placed in the regular breeding boxes but were moved into a dlff'erf.mt room wht6h did not have 75 automatic temperature regulation. Except for this change, the second colony was r,;i von exac tly the sane ca.re a s was given the first. '11he breeding animals were periodically inspected, pregnancies and births were recorded, the young were weaned, and the sexes were separated as previously described. At the time of weaning, all animals were marked and their birth dates were recorded. At'_di tional breeding animals were selec ted at random from among the young animals, but the rnatings were not between brothers and sisters. \Vhen a sufficient number of breeding animals was obtained, the offspring were placed in large wire cages in a separate room and designated as stock. The ca.':es in which the stock an~_mals were placed were constructed of sheet metal with a front and bottom of hardware cloth. The feeding and watering facilities in the metal cages were as adequate as those in the breeding boxes. Ten to fifteen nnimals were kept in each of thesE'. cages for over a year. The anbll.l.1s Vlere heal thy and mortality was very low. 'l'he animals in eaerl cage were of approximately the same age and thus it was?ossible to keep a close inventory of the number of animals and their ages at all times. When sufficient numbers of animals of the right ages were obtained the eX;1sriments were performed. Si~:ce it was desired tc eompatte the body and organ weights of males and females ranging from prepuberty to senility, the 8.[1;es of six, fourteen, twenty-six, thirty-two, and Sixty-two weeks were chosen. These represent the prepubertal (six weeks), pubertal (fourteen weeks), me ture{ twenty-six and thirty-two 76 weeks), and senile (sixty-two weeks) stages of life. At least eip,ht animals of each sex in each age class were used in this study. Total leulrocyte and differential blood counts were made 'before the ani~lla.lswerI"e sacrificed. Body and tissue weiFt,hts were obtained, s.nd the tissues wore fixed for histological study as previously described. The lymphatic organs sampled were: The spleens. thymi, rnesenteric, cervical, axil­lary, and inc~uinal nodes. In adliltion, the weights of both testes and adrenals in the males and the left ovary, uterine salpinx and cornu and both adrenals in the females were obtained. The ovary was too difficult to dissect out alone and too small for accurate weifr.hing •. In order to simplify the terminology, the ovary, sa.lpinx and cornu are collectively referred to as rlovary and tube", although. anatomically, this term is not accurate. 3. Results sta.tistical summa.ries of the data. obtained in thIs study are presented in Tables 8 and 9, which give the absolute and relative weights of the various or~ans in males and females. In this study the axillary, inguinal and cervical nodes were grouped together and designated as peripheral nodes. The mesenteric nodes Y/ere studied separately but were also grouped with tIle peri')herB,1 nodes and designated as total nodes. The catef:~ory, total lymphatic organs, ihrludes the spleens, thymi, mesenteric nodes, and,)sripb.eral nodes. An inspection of the statistical data shows that the standard errors were not large except for the spleens and total lympha tic organs. These large ~ Boq in Wt. W,k a. a. •• 17.27 .:t.26 14 25.7 -+.21 ~ 26 28.4 .:t.64 32 30.)2 .:t.43 62 29.67 .t.68 '6 15.2 .:t.l 14 20.1 . ~• 26 2-+2..276 «I l .:tl •S )2 24.8 ..t.59 62 24.16 +.5 77 Table 8 iiIean Body Weights and lkHl.n Absolute Weights ot Lynphatio Organs, Genital Organs, and Adrenals of CBA Mice Perl.- Me ..... fotal phera1 teric Total Iqm. !lu. III-I II'·· Sillen !9;pa 91aes Ooeada 15.8 22., 38.) 74.5 61.; 114.3 100.4 ,;tl.2 .t1•2 +1.3 ,;tlO.9 .t1•1 ill.) .t4.7 27.6 23.0 50.6 110.4 33.5 194.5 160.0 .:t2.4 .:t1•21 -+2.7 .112•2 .:t2•2 i 14.2 ..t2•S 2).6 16.2 39.8 109.5 18.1 lC8.0 161., -+1.84 .11•0 -+2* 1 -+8.8 .11.9 .;t.ll.O -+4.5 22.0 14.25 36.4 94.5 16.75 147.62 162.37 -+1.6 -+1.1 -+2.1 . :J:J.9 -+1.9 -+8.7 -+4.9 22.5 8.75 )1.1 87.81 ,.06 122.3 1)5.5 .;tl.2 .±l.O -+1.9 .115.3 ..:.t1•O .t10•8 .:t 2.7 18.2 21.1 39.3 61.4 66.5 167.4 11.7 .:t. '1 -+.7 .:t1•2 -+4.' ;t2.4 j;.6.0 .t2•0 23.6 24.3 47.' 110.6 37.1 196.0 32.1 ;tl.4 .11.2 -+2.2 -+4.2 -+1.' -+6" 8 .:th) 24.2 14.4 38.6 103.9 32., 176.0 53.2 ;tl.a .±.l.S .;tl.4 .:tIO.) z2.7 z.12.J .14.9 19.3 17.2 )8.3 98.7 29.4 164.5 57.2 ..t1.3 .±.2.0 .t3.3 -+8.6 .:tI •S -+11.7 j:4.9 16.68 19.0 35.7 71.12 20.5 133.31 60.6 j:l.3 ~.7 .14.7 .,:t4.0 -+1.6 -+6.9 .t18.1 Adr.- nals 2.8 1.' ..t.l ).2 -+ ••2 2.75 .±..48 2.1 .:t.OO2 ).1 ).8 .1.28 4.2 3.9 4.0 Age Body in Wi. Wka, Oms, 6 11.) .:t.26 14 25.7 -+.27 26 28.4 " .:t.64 ~ 32 30.3 .t.43 62 29.1 -+.68 6 15.2 .:t.3 14 20.1 26 .t. 26 C'O 26 22.7 0> 'it ! 32 24.8 .:t.o 62 24.2 .:t.5 78 Table 9 Mean Relative Weihts of LjT.lphatic Organs, Genital Organs, and Ad.renals oJ.' CBA M1.ce Peri~,.; ... n- 'otal ph.ral ter1:o '0 tal L,rt4. Nodee 1104 •• Nodes Si1een ~. J~ 0il&~. Gonad. 91.5 1)0.0 221.3 431 356.5 l008.S 581.3 101.6 89.1 197.3 430 130.5 758.0 62).0 83.0 57.0 140.0 385 65.6 590.1 568.0 12.0 41.0 120.0 311.9 55.3 487.1 489.0 15.8 29.5 104.8 295.9 30.6 412" 456.5 119.8 139.0 258., 404.0 437.6 1102 77 117.5 121.0 236.4 550.0 184.5 975 160 106.5 ·63.4 169.8 457.0 143.0 770 234 77.8 69.2 154.5 398.0 118.5 655 2)0 69.1 78.7 147.6 319.0 84.9 552 251 Aue-nal, 16.2 14.0 11.1 9.1 9.1 20.4 18.9 17.8 15.7 16.6 79 et&Ildard (!1rl~Ot"S were d.ue to the G~'O'lt varIabilIty 1n t1:~e welfr..hts of' the ~plG(lnl at all aq-tls. The$8 ~ta wero ft~!'th(;'r plotted hI Graphi0 form and r~ay be core easily Qnaly~ed by an (u::&:r:'flug,t1on ot ttl~ gr(}wth curvee 0.(. ' eaolI l OJ.... . i..-. ~l 1~ (''irgana S t tlAu~ ... $t;..~ ; (">.f4 -' mUNe 1~v t 0 I""'I''~'. ) • Ocneral bodyt"'t*Owth (F!'.' i;fJ1"f) l~ l.. At $1x W(i)~!·:g of' 1Afi\0' the I -,.. , _ u¥il 'OP'. 01 at U'I.' 1 ,:. D$:,"ond tl~f,r-t-::r ... tw·:' "0(,,\(1, tb:e })·;:,(11 ".l~ht C'JrVtHt leveled ~rr nnd tondod to <.1ro:,) G 11 Hht.17( by the tJlxtY-leC:h:md WfH,k. 30 20 ,/ 0/ 10 o 6 ,/-6' ----- /' 14 --___ -0-­...--- 0 --------- Age in Weaks. Pig. 15. Changes with Age in Body Weight of Male and Female CBA race. 62 • o SO The maximum difference in body weights between males and females wos attained by the fourteenth week. Beyond fourteen weeks, the~~rowth curves ran near-I:! parallel and the differ­ences between the two remainEHl nearly the same. Growth of tr'l6 testes (Figures 16 and 17). The maximum weiGht of the testes was reached at fourteen weeks, at which time the body weight was still increasing, but at a slower rate. Between fourteen and tLirty-two weeks the absolute· weight of the testes remained constant but after t:~irty-two weeks the weight dropped so that at sixty-two weeks the mean weight of the testes wa.s twenty-four milligrams b610w the maxhHlm of one hundred sixty milligrams. The changes in relative weights of the testes between six Ilnd fourteen weeks Yl'::re much less than changes in their ab­solute weights. This was oorrelated with the rapid rise in body wei t (;uring t·~ls perIod. Between the ages of fourteen and thirty-two weeks, there wore decreases in relative weights. T~esB were correlated with ~:e decrease in absolute Growth and wi th the increase in body weight, which was still taking place. The decrease in relative weight between thirty-two and sixty­two weeks WB.S due to a decrease in ah:;;olute weight c.uring the period in which the body weight was constant. Growth of the ovary: and ute~ine tube (Figures 16 and 17!) The growth of the ovary and uterln.e tube was much slower than that of the testes, and the mean maximum weight was not attained until the a.r~e of thirty-two weeks. Beyond that age UJ ~ H ffl or-! r-I r-I 'r-I ",:: ..... .,J.:.:, ~ .c M ....t (I) ~ • +J 200 100 0 tiO 30 0 ~ 600 ~ '1j o l:Q' ..r- , r' " 6 14 cr- - " 0" Fig. --- .- 81 --- Testes _~_----t- - ---- --- -- -----~ - - - Ovary & Uterine Horn 26 32 Age in Weeks 18. Absolute Growth of '3eni tal Ti.ssue Testes -- - --- .."J:J-- - - -0--- 62 _-0 - .... Ovary & Uterine Tube o~------~--------~~--~------------------------~ 6 14 26 32 Age in Weeks Fig. 17. Relative Growth of Genital Tissue. - 62 82 ~rowth virtually ceased. In comparing the growth curve of the ovary-uterine tube with the growtl1 curve of the testes, it must be e:-:lphasized that the uterine tube comprj.sed the greatest bulk of the fet.:lale gani tal ti s sue. The use of the Whole g;eni­tal tract ~;l[ly be further ,justified by the fact th.at the weight of tl:1e cornu changes directly wi th ovarian function. The shape of the growth curve of the ovary-uterine tube resembles that for body r;rowth, in that it levels off Ie' tel" the thirty-second week. Growth of the adre~lCl; land (Fit,;ure 18). Significant differences were observed between the absolute weights of the adre:-:all ,,,;lanc:s of males and fe:.-nales at all· ages. ~:he dlffer­encf: s were sna 11 a t six and fourteen weeks of age, but were p;rea tel' in the older anLnals. In female~ ,-tf.'owth of the adrenals continued u~) to twenty-six weeks of age, and then began to undeI'- go a slight decrease in weight. In ~ales, an increase in ab-solute wel t occurred only up to fourteen weeks and the sub­sequent decline in weight was e;rea ter than in females. ffhe sex differences in absolute wei gh ts 0 f the adrEnal n;lands resulted in the larQ;e sex differences in their rela.tive wei[",;hts. Although the relative weights were higher in females, progressive losses with a.:'o occur:rooa in both sexes during the whole period ooserved. 'l'hese losses were more rar)id during the first thirty-two weeks of life, but between thirty-two Bnd sJxtv-two weo\"s there were no further si(~ ~ ficant Chanr"6s. ... ...._ , •. t The losses in r 1ntive wei.ghts between the sixth and the I1J a C\l ~4 ~:') .,.-/ r-i r... l, ~,' ! ..c..:. .p .~..'..) (I) ~ fI) E as -4 3 2 1 IE 15 oo ~ • fI) S t,o 10 ~ !:l oM .p .c M oM ~ 5 6 - 0- / - 83 -- _0 ...... ' .... -, ..... .... 0- Absolute -- --- -"-0 M - -- F Relative " ..... _,,-- ______ --0 '00- - - 14 26 :32 Age in Weeks Fig. U:. Normal Growth and Involution of the Adrenal Glands 62 84 f01..U'teenth weeks in :nales and the sixth and t?Jent